Simultaneous multiplex genome editing in yeast

ABSTRACT

The present disclosure provides compositions of matter, methods and instruments for editing nucleic acids in live yeast cells.

RELATED CASES

The present application is a continuation of U.S. Ser. No. 17/463,956,filed 1 Sep. 2021, now allowed; which is a continuation of U.S. Ser. No.17/230,241, filed 14 Apr. 2021, now U.S. Pat. No. 11,136,572; which is acontinuation of U.S. Ser. No. 17/061,143, filed 1 Oct. 2020, now U.S.Pat. No. 11,001,831; which is a continuation-in-part of U.S. Ser. No.16/827,639, filed 23 Mar. 2020, now U.S. Pat. No. 10,815,467; whichclaims priority to U.S. Ser. No. 62/823,136, filed 25 Mar. 2019; U.S.Ser. No. 62/871,879, filed 9 Jul. 2019; and U.S. Ser. No. 62/960,291,filed 13 Jan. 2020.

FIELD OF THE INVENTION

This invention relates to compositions of matter, methods andinstruments for nucleic acid-guided nuclease editing of live yeastcells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise. targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently various nucleases have been identified that allowmanipulation of gene sequence, and hence gene function.

The nucleases include nucleic acid-guided nucleases, which enableresearchers to generate permanent edits in live cells. Editingefficiencies frequently correlate with the level of expression of guideRN As (gRNA s) in the cell. That is, the higher the expression level ofgRNA, the better the editing efficiency. Moreover, editing efficienciesin eukaryotes also correlate with the gRNAs being localized in thenucleus; that is, for efficient editing to occur, the gRNAs must remainin the nucleus to direct editing, rather than being exported from thenucleus to the cytoplasm. Additionally, it is desirable to minimize thenumber of editing rounds yet obtain a high number of edit in a cellulargenome; however, the architecture of many current editing systems onlyallows one edit per round

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved methods for increased transcription and nuclearlocalization of gRNAs, as well as methods and compositions forsimultaneous combinatorial editing. The present invention satisfies thisneed.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure relates to methods and compositions for makingseveral edits to a yeast genome simultaneously, as well as modules andautomated multi-module cell processing instruments configured to performthe methods utilizing the compositions.

In yeast, such as Saccharomyces cerevisiae (S. cerevisiae), gRNAexpression systems can use RNA polymerase III (pol III) or RNApolymerase II (pol II) promoters to drive transcription. The availablenumber of pol III promoters in yeast is limited as compared to pol IIpromoters. In general, pol III promoters have lower expression levelsthan pol II promoters and pol II promoters are more tolerant to sequencevariations than are pol III promoters, which allows for increasedsequence flexibility. In a multiplexed gene editing system, theexpression level of gRNAs is a critical factor in editing efficiency andefficacy. Pol II promoters generally express gRNAs that are subsequentlypoly-adenylated, marking the transcript for nuclear export; however, inorder to use pol II gRNA expression in a gene editing system, gRNAsexpressed from the pol II promoter must remain in the nucleus. Toaccomplish gRNA nuclear localization after expression from a pol IIpromoter, a self-cleaving ribozyme is added to the 3′ end of the gRNAtranscript. This self-cleaving ribozyme cleaves off the poly-A nuclearexport tag. In some embodiments, a self-cleaving ribozyme is also added5′ of the transcript to cleave off the post-transcriptionally added 5′cap, which also, when present, helps mediate nuclear export of mRNA.

Thus, in one embodiment there is provided a ribozyme-containing editingcassette for performing RNA-directed nuclease editing in yeastcomprising from 5′ to 3′: a pol II promoter, a transcription start site,a coding sequence for a gRNA, a coding sequence for a donor DNA, acoding sequence for a self-cleaving ribozyme, and a pol II terminator;or a pol II promoter, a transcription start site, a coding sequence fora donor DNA, a coding sequence for a gRNA, a coding sequence for aself-cleaving ribozyme, and a pol II terminator. That is, theribozyme-containing editing cassettes of the present invention areagnostic to the order of the gRNA and donor DNA coding sequences in theribozyme-containing editing cassette.

In addition, in preferred aspects the ribozyme-containing editingcassettes comprise regions of homology to a vector backbone forgap-repair insert of the ribozyme-containing editing cassettes into thevector backbone.

Further, in some aspects, the ribozyme-containing editing cassettefurther comprises a second self-cleaving ribozyme 3′ of thetranscription start site.

In some aspects of this embodiment, the self-cleaving ribozyme of theribozyme-containing editing cassette is selected from a self-cleavingribozyme in a hepatitis delta virus (HDV)-like ribozyme family, aself-cleaving ribozyme in a glucosamine-6-phosphate synthase ribozymefamily, a self-cleaving ribozyme in a hammerhead ribozyme family, aself-cleaving ribozyme in a hairpin ribozyme family, a self-cleavingribozyme in a Neurospora Varkud satellite ribozyme family, aself-cleaving ribozyme in a twister ribozyme family, a self-cleavingribozyme in a twister sister ribozyme family, a self-cleaving ribozymein a hatchet ribozyme family, or a self-cleaving ribozyme in a pistolribozyme family. Additionally, in some aspects of this embodiment, thereis a second self-cleaving ribozyme sequence located 3′ of thetranscription start site and 5′ of the first coding sequence for anucleic acids-guided editing component (e.g., either the gRNA or donorDNA) of the editing cassette.

Also, in some embodiments, the pol II promoter of the composition is acell-type specific promoter, a tissue-specific promoter, or a syntheticpromoter. In some aspects, the pol II promoter is a constitutive fungalpromoter, and in some aspects, the constitutive fungal pol II promoteris selected from a pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2,pYEF3, pRPL3, pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, or pPDA1 promoter.In yet other aspects, the pol II promoter may be a constitutivemammalian promoter, such as the pCMV, pEF1a, pSV40, pPGK1, pUbc, humanbeta actin promoter, or pCAG promoter. Alternatively, the pol IIpromoter may be an inducible promoter, such as the PHO5 promoter, theMET3 promoter, the CUP1 promoter, the GAL1 promoter, or the GEV or LEVpromoter system.

Yet other embodiments provide a method for RNA-directed nuclease editingin yeast cells comprising the steps of: designing and synthesizingribozyme-containing editing cassettes, wherein the ribozyme-containingediting cassettes comprise from 5′ to 3′: 1) a pol II promoter, atranscription start site, a coding sequence for a gRNA, a codingsequence for a donor DNA, a coding sequence for a self-cleavingribozyme, and a pol II terminator, or 2) a pol II promoter, atranscription start site, a coding sequence for a donor DNA, a codingsequence for a gRNA, a coding sequence for a self-cleaving ribozyme, anda pol II terminator; amplifying the synthesized ribozyme-containingediting cassettes; transforming the yeast cells with theribozyme-containing editing cassettes and vector backbone; selecting fortransformed yeast cells; providing conditions for RNA-directed nucleaseediting; and using the edited yeast cells in research.

In some aspects of the method, after the second providing step (e.g.,providing conditions for RNA-directed nuclease editing), a secondselecting step is performed thereby selecting for edited cells; and insome aspects of the methods, the designing, amplifying, first providing,transforming, first selecting, second providing and second selectingsteps are repeated until a desired number of edits have been made to theyeast cells.

In other embodiments there is provided a dual ribozyme-containingediting cassette for performing RNA-directed nuclease editing in yeastcomprising from 5′ to 3′: a pol II promoter; a transcription start site;a first editing cassette, wherein the editing cassette comprises acoding sequence for a first gRNA and a coding sequence for a first donorDNA, and wherein the first donor DNA comprises a rational, desired editto a first target sequence and an edit configured to render inactive aproto-spacer motif (PAM) in the first target sequence; a linker orspacer (not to be confused with a proto-spacer motif (PAM)); a secondediting cassette (e.g., here a ribozyme-containing editing cassette),wherein the second editing cassette comprises a coding sequence for asecond gRNA and a coding sequence for a second donor DNA, wherein thesecond donor DNA comprises a rational, desired edit to a second targetsequence and an edit configured to render inactive a proto-spacer motif(PAM) in the second target sequence, a coding sequence for aself-cleaving ribozyme, and a pol II terminator.

In some aspects, the dual ribozyme-containing editing cassette furthercomprises a second self-cleaving ribozyme 3′ of the transcription startsite. In some aspects, the linker or spacer is a coding sequence for atRNA whereas in other aspects, the linker or spacer is a primersequence. In some embodiments, the ribozyme-containing editing cassettefurther comprises between the second editing cassette and the codingsequence for a self-cleaving ribozyme a second linker or spacer and athird editing cassette, wherein the third editing cassette comprises acoding sequence for a third gRNA and a coding sequence for a third donorDNA, and wherein the third donor DNA comprises a rational, desired editto a third target sequence and an edit configured to render inactive aproto-spacer motif (PAM) in the third target sequence thus resulting ina multiplex ribozyme-containing editing cassette. In some aspects, amultiplex ribozyme-containing editing cassette may comprise a fourth,fifth and even sixth editing cassette where each of the fourth, fifthand sixth editing cassettes are separated from the other editingcassettes by a linker or spacer.

In some aspects, the coding sequence for the first gRNA is 5′ of thecoding sequence of the first donor DNA, whereas in other aspects, thecoding sequence for the first gRNA is 3′ of the coding sequence of thefirst donor DNA. In some aspects, the coding sequence for the secondgRNA is 5′ of the coding sequence of the second donor DNA, whereas inother aspects, the coding sequence for the second gRNA is 3′ of thecoding sequence of the second donor DNA. Again, the ribozyme-containingediting cassettes (and editing cassettes) of the present invention areagnostic to the order of the gRNA and donor DNA coding sequences in theribozyme-containing editing cassette.

In yet another embodiment there is provided a library of linear vectorbackbones and a library of the editing cassettes or theribozyme-containing editing cassettes to be transformed into yeast cellscomprising: a first linear vector backbone comprising a coding sequencefor a nuclease, a coding sequence for a first antibiotic resistancegene, and a 2 origin of replication; and a library of editing cassettesor ribozyme-containing editing cassettes, wherein the gRNAs and donorDNAs of different editing cassettes or ribozyme-containing editingcassettes in the library target different target regions in a yeastgenome, and wherein homology exists between the library of editingcassettes or ribozyme-containing editing cassettes and the first linearvector.

In some aspects, there is also provided a library of linear vectorbackbones and a library of editing cassettes or ribozyme-containingediting cassettes to be transformed into yeast cells comprising: a firstlinear vector backbone comprising a coding sequence for a nuclease, acoding sequence for a first antibiotic gene, and a 2μ origin ofreplication; a second linear vector backbone comprising a codingsequence for the nuclease, a coding sequence for a second antibioticresistance gene, and a 2μ origin of replication; and a library ofediting cassettes or the ribozyme-containing editing cassettes, whereinthe gRNAs and donor DNAs of different editing cassettes orribozyme-containing editing cassettes in the library target differenttarget regions in a yeast genome, and wherein homology exists betweenthe library of editing cassettes or ribozyme-containing editingcassettes and the first and second linear vectors.

In some aspects of this embodiment, the library of linear vectorbackbones further comprises a third linear vector backbone comprising acoding sequence for the nuclease, a coding sequence for a thirdantibiotic resistance gene, and a 2μ origin of replication; and in someaspects, the library of linear vector backbones further comprises afourth linear vector backbone comprising a coding sequence for thenuclease, a coding sequence for a fourth antibiotic resistance gene, anda 2μ origin of replication and even a fifth linear vector backbonecomprising a coding sequence for the nuclease, a coding sequence for afifth antibiotic resistance gene, and a 2μ origin of replication. Insome aspects, the coding sequence for the nuclease in each of the first,second, third, fourth and fifth linear vectors is the same codingsequence, however in other aspects, the coding sequence for the nucleasein each of the first, second, third, fourth and fifth linear vectors maybe a different coding sequence.

In some aspects, the first antibiotic resistance gene confers resistanceto hygromycin and the second antibiotic resistance gene confersresistance to G418.

In some aspects, each editing cassette or ribozyme-containing editingcassette comprises two gRNAs and two donor DNAs, or three gRNAs andthree donor DNAs.

In some aspects, each linear vector backbone comprises a promoterpositioned to drive transcription of the editing cassette such as a polII promoter, and in some aspects each linear vector backbone furthercomprises an origin of replication functional in bacteria. That is, insome aspects the promoter driving transcription of the editing cassetteis on the vector backbone rather than including in the editing cassetteor ribozyme-containing editing cassette.

Other embodiments provide a method of performing nucleic acid guidednuclease editing in yeast cells comprising the steps of: 1) transformingyeast cells with a library of linear vector backbones and a library ofediting cassettes or ribozyme-containing editing cassettes to betransformed into yeast cells wherein the library of linear vectorbackbones comprises: a first linear vector backbone comprising a codingsequence for a nuclease, a coding sequence for a first antibioticresistance gene, and a 2μ origin of replication; a second linear vectorbackbone comprising a coding sequence for the nuclease, a codingsequence for a second antibiotic resistance gene, and a 2μ origin ofreplication; and a library of editing cassettes or ribozyme-containingediting cassettes, wherein each editing cassette comprises a gRNA and adonor DNA, wherein the gRNAs and donor DNAs of different cassettestarget different target regions in a yeast genome, and wherein homologyexists between the library of editing cassettes and the first and secondlinear vectors; 2) selecting for yeast cells resistant to the first andsecond antibiotic resistance genes; 3) allowing the yeast cells torecover and for the nucleic acid-guided nuclease editing to take place;and 4) growing the edited yeast cells to stationary phase. Once theyeast cells have been grown to stationary phase, the cells can then bepooled and rendered electrocompetent for another round of editing. Insome aspects of this embodiment, the yeast cells may be singulated afterthe transformation step.

Yet another embodiment provides a library of linear vector backbones anda library of editing cassettes or ribozyme-containing editing cassettesto be transformed into yeast cells comprising: a first linear vectorbackbone comprising a coding sequence for a nuclease, a coding sequencefor a first antibiotic resistance gene, and a 2μ origin of replication;and a second linear vector backbone comprising a coding sequence for thenuclease, a coding sequence for a second antibiotic resistance gene, anda 2μ origin of replication; and a library of editing cassettes orribozyme-containing editing cassettes, wherein each editing cassette orribozyme-containing editing cassette comprises a gRNA and a donor DNA,wherein the gRNAs and donor DNAs of different editing cassettes orribozyme-containing editing cassettes target different target regions ina yeast genome, and wherein homology exists between the library ofediting cassettes or ribozyme-containing editing cassette and the linearvector backbones.

In other embodiments there is provided a composition of mattercomprising libraries of linear vector backbones and libraries of editingcassettes to be transformed into yeast cells comprising: a first linearvector backbone library comprising a coding sequence for a nuclease, acoding sequence for a first antibiotic resistance gene, first homologyregions for inserting a first library of editing cassettes, and a 2μorigin of replication; a second linear vector backbone librarycomprising a coding sequence for a nuclease, a coding sequence for asecond antibiotic resistance gene, second homology regions for insertinga second library of editing cassettes, and a 2μ origin of replication;the first library of editing cassettes, wherein each editing cassettecomprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs ofdifferent cassettes target different target regions in a yeast genome,and wherein homology exists between the first library of editingcassettes and the first linear vector library; and the second library ofediting cassettes, wherein each editing cassette comprises a gRNA and adonor DNA, wherein the gRNAs and donor DNAs of different cassettestarget different target regions in a yeast genome, and wherein homologyexists between the second library of editing cassettes and the secondlinear vector library.

In some aspects of this embodiment, the composition of matter furthercomprises a third linear vector backbone library comprising a codingsequence for a nuclease, third homology regions for inserting a thirdlibrary of editing cassettes, a coding sequence for a third antibioticresistance gene, and a 2μ origin of replication; and the third libraryof editing cassettes, wherein each editing cassette comprises a gRNA anda donor DNA, wherein the gRNAs and donor DNAs of different cassettestarget different target regions in a yeast genome, and wherein homologyexists between the third library of editing cassettes and the thirdlinear vector library. In some aspects, the composition furthercomprises a fourth linear vector backbone library comprising a codingsequence for a nuclease, fourth homology regions for inserting a fourthlibrary of editing cassettes, a coding sequence for a fourth antibioticresistance gene, and a 2μ origin of replication; and the fourth libraryof editing cassettes, wherein each editing cassette comprises a gRNA anda donor DNA, wherein the gRNAs and donor DNAs of different cassettestarget different target regions in a yeast genome, and wherein homologyexists between the fourth library of editing cassettes and the fourthlinear vector library.

Yet another embodiment provides a composition of matter comprisinglibraries of linear vector backbones and libraries of editing cassettesto be transformed into yeast cells comprising: a first linear vectorbackbone library comprising a coding sequence for a nuclease, a codingsequence for a first portion of a first antibiotic resistance gene fusedto an N-terminus of a first intein, first homology regions for insertinga first library of editing cassettes, and a 2μ origin of replication; asecond linear vector backbone library comprising a coding sequence for anuclease, a coding sequence for a second portion of a first antibioticresistance gene fused to an C-terminus of the first intein, secondhomology regions for inserting a second library of editing cassettes,and a 2μ origin of replication; the first library of editing cassettes,wherein each editing cassette comprises a gRNA and a donor DNA, whereinthe gRNAs and donor DNAs of different cassettes target different targetregions in a yeast genome, and wherein homology exists between the firstlibrary of editing cassettes and the first linear vector library; andthe second library of editing cassettes, wherein each editing cassettecomprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs ofdifferent cassettes target different target regions in a yeast genome,and wherein homology exists between the second library of editingcassettes and the second linear vector library.

In some aspects of this embodiment the composition of matter of claimfurther comprises a third linear vector backbone library comprising acoding sequence for a nuclease, third homology regions for inserting athird library of editing cassettes, a coding sequence for a firstportion of a second antibiotic resistance gene fused to the N-terminusof a second intein, and a 2μ origin of replication; a fourth linearvector backbone library comprising a coding sequence for a nuclease,fourth homology regions for inserting a fourth library of editingcassettes, a coding sequence for a second portion of a second antibioticresistance gene fused to a C-terminus of the second intein, and a 2μorigin of replication; the third library of editing cassettes, whereineach editing cassette comprises a gRNA and a donor DNA, wherein thegRNAs and donor DNAs of different cassettes target different targetregions in a yeast genome, and wherein homology exists between the thirdlibrary of editing cassettes and the third linear vector library; andthe fourth library of editing cassettes, wherein each editing cassettecomprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs ofdifferent cassettes target different target regions in a yeast genome,and wherein homology exists between the fourth library of editingcassettes and the fourth linear vector library.

In some aspects of this embodiment, the first intein is derived fromNostoc punctiforme PCC73102 split alpha subunit of the DNA polymeraseIII intein (NpuDnaE), Synechocystis sp. PCC6803 DnaB helicase SspDnaB,or CfaDnaE (a consensus alignment of Npu and Ssp).

In some aspects of these embodiments of compositions, the first andsecond antibiotic resistance genes confer resistance to hygromycin,G418, puromycin, blasticidin or nourseothricin and the first and secondantibiotic resistance genes are different.

In some aspects of these embodiments of compositions, each editingcassette of each library of editing cassettes comprises two gRNAs andtwo donor DNAs, and in some aspects, each editing cassette of eachlibrary of editing cassettes comprises three gRNAs and three donor DNAs.

In some aspects of these compositions, each linear vector backbone ineach linear backbone library further comprises a promoter drivingexpression of the editing cassette, and in some aspects the promoter isa pol II promoter.

In some aspects of these embodiments of compositions, wherein eachlinear vector backbone in each linear backbone library further comprisesan origin of replication functional in bacteria.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is a simplified block diagram of an exemplary method for editinglive yeast cells via nucleic acid-guided nuclease editing. FIG. 1B is agraphic depiction of an exemplary embodiment of a ribozyme-containingediting cassette for nucleic acid-guided nuclease editing. FIG. 1C is agraphic depiction of an exemplary embodiment of a dualribozyme-containing editing cassette for nucleic acid-guided nucleaseediting. FIG. 1D is a simplified graphic of a method for editing yeastgenomes using a single vector backbone and a library of editingcassettes, and adjusting the concentration of the editing cassettes inthe transformation reaction to drive formation of more than one editingvector per cell to achieve two to many edits per cell per round. FIG. 1Eis a simplified graphic of an alternative system for editing yeastgenomes with two to many edits per round of editing using two differentvector backbones. FIG. 1F (i) shows the result of multi-vectortransformation without selective homology (as in the last panel of FIG.1E) and FIG. 1F (ii) shows the result of multi-vector transformationwith selective homology. FIG. 1G is a simplified graphic of analternative system for editing yeast genomes with two to many edits perround of editing using two different vector backbones, each with aportion of a coding sequence for an antibiotic marker. FIG. 1H is anexemplary map for a vector backbone for performing multiplexsimultaneous nucleic acid-guided nuclease editing.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing employing a split protein reporter system.

FIG. 3A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein and in relation to FIGS. 3B-3D.FIG. 3B illustrates a perspective view of one embodiment of a rotatinggrowth vial in a cell growth module housing. FIG. 3C depicts a cut-awayview of the cell growth module from FIG. 3B. FIG. 3D illustrates thecell growth module of FIG. 3B coupled to LED, detector, and temperatureregulating components.

FIG. 4A depicts retentate (top) and permeate (bottom) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom). FIG. 4B depicts twoside perspective views of a reservoir assembly of a tangential flowfiltration module. FIGS. 4C-4E depict an exemplary top, with fluidic andpneumatic ports and gasket suitable for the reservoir assemblies shownin FIG. 4B.

FIG. 5A depicts an exemplary combination reagent cartridge andelectroporation device (e.g., transformation module) that may be used ina multi-module cell processing instrument. FIG. 5B is a top perspectiveview of one embodiment of an exemplary flow-through electroporationdevice that may be part of a reagent cartridge. FIG. 5C depicts a bottomperspective view of one embodiment of an exemplary flow-throughelectroporation device that may be part of a reagent cartridge. FIGS.5D-5F depict a top perspective view, a top view of a cross section, anda side perspective view of a cross section of an FTEP device useful in amulti-module automated cell processing instrument such as that shown inFIGS. 2A-2C.

FIG. 6A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells in a solid wall device. FIGS. 6B-6D depictan embodiment of a solid wall isolation incubation and normalization(SWIIN) module. FIG. 6E depicts the embodiment of the SWIIN module inFIGS. 6B-6D further comprising a heater and a heated cover.

FIG. 7 is a simplified process diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument.

FIG. 8 is a simplified process diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module for recursive yeastcell editing.

FIG. 9 is a graph demonstrating real-time monitoring of growth of S.cerevisiae yeast cell culture OD₆₀₀ employing the cell growth device asdescribed in relation to FIGS. 3A-3D where a 2-paddle rotating growthvial was used.

FIG. 10 is a graph plotting filtrate conductivity against filterprocessing time for a yeast culture processed in the cell concentrationdevice/module described in relation to FIGS. 4A-4E.

FIG. 11 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device as described in relation to FIGS. 5A-5Fand a comparator electroporation method.

FIG. 12 shows the results of multiplex editing observed withribozyme-containing editing cassettes.

FIG. 13 shows the edit rates obtained for each gene targeted for editsby dual ribozyme-containing editing cassettes tested in yeast.

FIG. 14 shows the results from using a single vector backbone andediting cassette library to simultaneously edit multiple loci in thegenome of S. cerevisiae.

FIG. 15 shows the number of unique plasmids observed via NextGensequencing of a 500-member editing library across different libraryconcentrations.

FIG. 16 comprises two bar graphs showing the increased editing rateobtained utilizing a double antibiotic selection scheme versus a singleantibiotic selection scheme.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentare intended to be applicable to the additional embodiments describedherein except where expressly stated or where the feature or function isincompatible with the additional embodiments. For example, where a givenfeature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry and sequencing technology, whichare within the skill of those who practice in the art. Such conventionaltechniques include polymer array synthesis, hybridization and ligationof polynucleotides, and detection of hybridization using a label.Specific illustrations of suitable techniques can be had by reference tothe examples herein. However, other equivalent conventional procedurescan, of course, also be used. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Green,et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols.I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: ALaboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Bioinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3^(rd) Ed., W. H. Freeman Pub., New York,N.Y.; Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York, N.Y.; all of which are herein incorporated in their entiretyby reference for all purposes. CRISPR-specific techniques can be foundin, e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligonucleotide”refers to one or more oligonucleotides, and reference to “an automatedsystem” includes reference to equivalent steps and methods for use withthe system known to those skilled in the art, and so forth.Additionally, it is to be understood that terms such as “left,” “right,”“top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,”“upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may beused herein merely describe points of reference and do not necessarilylimit embodiments of the present disclosure to any particularorientation or configuration. Furthermore, terms such as “first,”“second,” “third,” etc., merely identify one of a number of portions,components, steps, operations, functions, and/or points of reference asdisclosed herein, and likewise do not necessarily limit embodiments ofthe present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents-translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” or “homologyarm” refers to nucleic acid that is designed to introduce a DNA sequencemodification (insertion, deletion, substitution) into a locus byhomologous recombination using nucleic acid-guided nucleases. Forhomology-directed repair, the donor DNA must have sufficient homology tothe regions flanking the “cut site” or site to be edited in the genomictarget sequence. The length of the homology arm(s) will depend on, e.g.,the type and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the target sequence.

The term “editing cassette” refers to a nucleic acid molecule comprisinga coding sequence for transcription of a guide nucleic acid or gRNAcovalently linked to a coding sequence for transcription of a donor DNAor homology arm. The term “ribozyme-containing editing cassette” refersto a nucleic acid molecule comprising an editing cassette and at leastone self-cleaving ribozyme.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.

Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Nucleic acid-guided editing components” refers to one, some, or all ofa nuclease, a guide nucleic acid, a donor nucleic acid, andrecombination systems, if required.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “PAM mutation” refers to one or more edits to a target sequence thatremoves, mutates, or otherwise renders inactive a PAM or spacer regionin the target sequence.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA. Promoters may be constitutive or inducible. A “pol II promoter” isa regulatory sequence that is bound by RNA polymerase II to catalyze thetranscription of DNA.

As used herein, a “ribozyme” (ribonucleic acid enzyme) is an RNAmolecule capable of catalyzing biochemical reactions. A “self-cleavingribozyme” is a ribozyme capable of cleaving itself.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, nourseothricin N-acetyl transferase,erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,puromycin, hygromycin, blasticidin, and G418 may be employed. In otherembodiments, selectable markers include, but are not limited to humannerve growth factor receptor (detected with a MAb, such as described inU.S. Pat. No. 6,365,373); truncated human growth factor receptor(detected with MAb); mutant human dihydrofolate reductase (DHFR;fluorescent MTX substrate available); secreted alkaline phosphatase(SEAP; fluorescent substrate available); human thymidylate synthase (TS;confers resistance to anti-cancer agent fluorodeoxyuridine); humanglutathione S-transferase alpha (GSTA1; conjugates glutathione to thestem cell selective alkylator busulfan; chemoprotective selectablemarker in CD34+ cells); CD24 cell surface antigen in hematopoietic stemcells; human CAD gene to confer resistance toN-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2α; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); rhamnose; and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome or episome) of a cell or population of cells, inwhich a change of at least one nucleotide is desired using a nucleicacid-guided nuclease editing system. The target sequence can be agenomic locus or extrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, BACs, YACs, PACs, synthetic chromosomes, andthe like.

Improved Nucleic Acid-Guided Nuclease Editing in Yeast

The present disclosure provides compositions of matter, methods andinstruments for nucleic acid-guided nuclease editing of live yeastcells, and in particular, high-throughput methods for increasing editingrates and allowing for multiplex simultaneous editing in live yeastcells. The compositions and methods described herein improve CRISPRediting systems in which nucleic acid-guided nucleases (e.g., RNA-guidednucleases) are used to edit specific target regions in a yeast genome. Anucleic acid-guided nuclease complexed with an appropriate syntheticguide nucleic acid in a yeast cell can cut the genome of the cell at adesired location. The guide nucleic acid helps the nucleic acid-guidednuclease recognize and cut the DNA at a specific target sequence. Bymanipulating the nucleotide sequence of the guide nucleic acid, thenucleic acid-guided nuclease may be programmed to target any DNAsequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length. The guide nucleic acid also comprises a scaffoldsequence capable of interacting or complexing with a nucleic acid-guidednuclease.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid or homology arm. The donor nucleic acid is on thesame polynucleotide (e.g., editing cassette or ribozyme-containingediting cassette) as the guide nucleic acid and typically is under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid) (see, e.g., FIGS. 1B, 1C and 1H). The donornucleic acid is designed to serve as a template for homologousrecombination with a target sequence nicked or cleaved by the nucleicacid-guided nuclease as a part of the gRNA/nuclease complex. A donornucleic acid polynucleotide may be of any suitable length, such as aboutor more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000nucleotides in length. In certain preferred aspects, the donor nucleicacid can be provided as an oligonucleotide of between 20-300nucleotides, more preferably between 50-250 nucleotides. The donornucleic acid comprises a region that is complementary to a portion ofthe target sequence (e.g., a homology arm). When optimally aligned, thedonor nucleic acid overlaps with (is complementary to) the targetsequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or morenucleotides. In many embodiments and preferably, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

The donor nucleic acid is provided—along with the gRNA (both scaffoldand guide sequences)—as one component in an editing cassette orribozyme-containing editing cassette, where the editing cassette orribozyme-containing editing cassette may be one of multiple editingcassettes inserted into a vector backbone. That is, there may be morethan one, e.g., two, three, four, five or more individual gRNA/donor DNApairs inserted into a vector backbone, where the multiple guide nucleicacid/donor nucleic acid pairs are under the control of a singlepromoter—in some embodiments, a pol II promoter, and in someembodiments, the promoter driving transcription of the gRNAs is aninducible pol II promoter. In some embodiments, transcription of thenuclease is also inducible; thus, in some embodiments, transcription ofboth the nuclease and gRNA are inducible. Inducible editing isadvantageous in that cells can be grown for several to many celldoublings to a stationary growth phase (or nearly so) before editing isinitiated, which increases the likelihood that cells with edits willsurvive. In some aspects, there are linker or spacer sequencesseparating the individual gRNA/donor DNA pairs from one another.

The guide nucleic acid can be engineered to target a desired targetsequence by altering the guide sequence so that the guide sequence iscomplementary to a desired target sequence, thereby allowinghybridization between the guide sequence and the target sequence. Ingeneral, to generate an edit in the target sequence, the gRNA/nucleasecomplex binds to a target sequence as determined by the guide RNA, andthe nuclease recognizes a roospacr adjacet (PAM) sequence adjacent tothe target sequence. The target sequence can be any genomic or episomicpolynucleotide whether endogenous or exogenous to a prokaryotic oreukaryotic cell, or in vitro. For example, the target sequence can be apolynucleotide residing in the nucleus of a yeast cell. A targetsequence can be a sequence encoding a gene product (e.g., a protein) ora non-coding sequence (e.g., a regulatory polynucleotide, an intron, aPAM, a spacer, or “junk” DNA).

The target sequence is associated with a protospacer adjacent motif(PAM), which is a short nucleotide sequence recognized by thegRNA/nuclease complex. The precise PAM sequence and length requirementsfor different nucleic acid-guided nucleases vary; however, PAMstypically are 2-7 base-pair sequences adjacent or in proximity to thetarget sequence and, depending on the nuclease, can be 5′ or 3′ to thetarget sequence. Thus, the editing cassette provides a donor DNAsequence (e.g., a homology arm) that, in addition to allowing forprecise genome editing of a target sequence also provides one or morechanges to the target sequence that removes, mutates or renders inactivethe proto-spacer adjacent motif (PAM) in the target sequence. Renderingthe PAM at the target sequence inactive precludes additional editing ofthe cell genome at that target sequence, e.g., upon subsequent exposureto a nucleic acid-guided nuclease complexed with a synthetic guidenucleic acid in later rounds of editing. Thus, cells having the desiredtarget sequence edit and an altered PAM can be selected using a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acidcomplementary to the target sequence. Cells that did not undergo thefirst editing event will be cut rendering a double-stranded DNA break,and thus will not continue to be viable. The cells containing thedesired target sequence edit and PAM alteration will not be cut, asthese edited cells no longer contain the necessary PAM site and willcontinue to grow and propagate.

Methods and compositions for designing and synthesizing editingcassettes are described in U.S. Pat. Nos. 10,240,167; 10,266,849;9,982,278; 10,351,877; 10,364,442; and 10,435,715; and U.S. Ser. No.16/275,465, filed 14 Feb. 2019, all of which are incorporated byreference herein. Again, each editing cassette and ribozyme-containingediting cassette comprises a gRNA sequence to be transcribed and a donorDNA or homology arm sequence to be transcribed, including both a desirededit and a PAM or spacer mutation. In the case of ribozyme-containingediting cassettes, at least one editing cassette is linked to a codingsequence for one or more self-cleaving ribozymes. Note that although thegRNA is shown 5′ to the donor DNA in FIGS. 1B and 1C, the gRNA could bepositioned 3′ of the donor DNA.

In certain of the present methods and compositions, the polynucleotidesequence encoding the editing cassette is linked to one or moreself-cleaving ribozyme sequences creating a ribozyme-containing editingcassette. The ribozyme sequence may be 20-200 nucleotides in length,more preferably 50-150 nucleotides in length. When transcribed to RNA,self-cleaving ribozyme sequences mediate site-specific phosphodiestercleavage of the transcript within or proximal to the ribozyme sequenceitself. Cleavage proceeds via general acid—general base catalysis,beginning with abstraction of a proton from the 2′ OH of the nucleotide5′ of the scissile bond. The resulting 2′ oxygen attacks the adjacent 3′phosphate, yielding a 2′-3′ cyclic phosphate in the upstream nucleotideand a 5′-hydroxyl product downstream of the scissile bond.

Ribozymes mediate sequence-specific cleavage of the internalphosphodiester bond by means of their unique secondary and tertiarystructure that organize after transcription to RNA, includingmultihelical junctions, interactions of nonhelical elements such ashelix-terminal loops and internal bulges, and pseudoknotting. Thesetertiary structures position the substrate—the scissile bond to becleaved-inside an active-site cleft surrounded by nucleotides that maybe distant in the primary sequence.

There are several families of ribozymes, each with unique structure andactive site conformation, but they all accomplish the same reaction ofsequence-specific phosphodiester cleavage. In the present methods andcompositions, the self-cleaving ribozyme sequence may be chosen from oneof hepatitis delta virus (HDV)-like, glucosamine-6-phosphate synthase(glmS), Neuropsora Varkud satellite (VS), hammerhead, twister, twistersister, hatchet, pistol, among others.

In some embodiments, the ribozyme sequence may be placed 3′ of thehomology arm of the editing cassette and 5′ of the pol II terminatorsequence (discussed below and shown in FIG. 1B), i.e. between thehomology arm and the pol II terminator sequence. In native contexts, thepol II terminator sequence typically coordinates termination andpolyadenylation, followed by nuclear export. In preferred embodiments ofthe present disclosure the ribozyme sequence mediates cleavage of thepoly(A) tail from the 3′ end of the transcript and prevents nuclearexport of the pol II-transcribed gRNA transcript. In some embodiments,two different self-cleaving ribozymes may be used with one ribozymeplaced at the 3′ end of the donor DNA or homology arm and with oneribozyme placed at the 5′ end of the editing cassette, 3′ of thetranscription start site. The 5′ ribozyme removes thepost-transcriptionally-added 5′ cap, thus further increasing thelikelihood the cassette transcript will be retained within the nucleus.

In a one embodiment, one ribozyme is HDV-like and the HDV-like ribozymeis placed 3′ of the homology arm or homology arms of the gRNA/donor DNApair and 5′ of the pol II terminator sequence, i.e., between thehomology arm and the pol II terminator sequence. The HDV-like ribozymemediates cleavage 5′ of the ribozyme sequence itself, minimizing theamount of HDV-like sequence remaining on the cleaved transcript. Also,in one embodiment, a hammerhead ribozyme is used 5′ of the editingcassette and 3′ of the promoter. Although here the choice ofself-cleaving ribozymes to be positioned 3′ of the transcription startsite and positioned 3′ of the editing cassette are hammerhead andHDV-like respectively, other arrangements can be used; for example, theself-cleaving ribozyme positioned 3′ of the transcription start site and5′ of the editing cassette may be an HDV-like ribozyme and theself-cleaving ribozyme positioned 3′ of the editing cassette and 5′ ofthe pol II terminator sequence may be a hammerhead ribozyme.Alternatively, both self-cleaving ribozymes may be the sameself-cleaving ribozyme such as two hammerhead ribozymes, though thisconfiguration is not preferred due to concerns of secondary structureformation and/or recombination between sites. In yet anotheralternative, any self-cleaving ribozyme may be used in either position,including—in addition to the HDV-like and hammerheadribozymes—glucosamine-6-phosphate synthase (glmS) ribozymes, NeuropsoraVarkud satellite (VS) ribozymes, twister ribozymes, twister sisterribozymes, hatchet ribozymes, and pistol ribozymes.

The ribonucleoprotein editing complex performs editing of its targetsequence in the nucleus; thus, the gRNA transcript must remain in thenucleus for editing to occur. In certain embodiments of the presentmethods and compositions, the guide nucleic acid and donor DNA areprovided as coding sequences in an editing cassette to be expressed froma plasmid or vector under the control of a pol II promoter. Previously,RNAs transcribed by an RNA pol II promoter could not be used as gRNAs,as they undergo significant post-transcriptional processing and nuclearexport. Therefore, RNA polymerase III promoters, e.g. U6, U3, U2, SNR52,RPR1, among others, have been used in the art to drive expression ofgRNAs. Pol III-expressed RNAs are not polyadenylated or exported fromthe nucleus. However, pol III promoters have several limitations. First,in the yeast genome for example, there are relatively few pol IIIpromoters available and they are generally lower-expressing than pol IIpromoters, limiting the dynamic range and expression levels availablefor gRNAs. Second, pol III promoters are not amenable to sequencealteration, e.g., the U6 promoter requires a guanine nucleotide at itstranscription initiation site, limiting gRNA target sequence selection.Additionally, pol III promoters are restrictive in that many of pol IIIpromoters have intragenic regulatory regions, such that changing thesequence to be expressed is not possible. Also, pol III genes aregenerally housekeeping genes and are constitutively and ubiquitouslyexpressed, precluding the use of inducible, cell-type, ortissue-specific promoters for gRNA expression. Finally, because thedonor DNA sequences (e.g., homology arm sequences) are typically derivedfrom genomic sequences, the donor DNA sequences may contain pol IIItermination motifs such as Penta T or Penta T+G. Termination motifs suchas these may prevent transcription of the functional gRNA portion of theediting cassettes.

In contrast, RNA pol II promoters are generally higher expressing andprovide a much larger dynamic range of possible expression levels ofgRNA compared to the limited number of pol III promoters. Pol IIpromoters are more amenable to sequence alteration and do not have thelimited sequence requirements of pol III promoters, thereby expandingthe editing space and flexibility of target sequences. Expressing gRNAswith pol II enables the use of the many cell-type specific, tissuespecific, or synthetic pol II promoters that have been designed,including inducible promoters, discussed below.

In some embodiments, the pol II promoter driving gRNA expression may beselected from one of many constitutive fungal promoters, including butnot limited to, pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2, pYEF3,pRPL3, pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, or pPDA1. As discussedabove, pol II promoters have a wide dynamic range of expression levels.In a preferred embodiment, gRNA expression may be driven by a pol IIpromoter known to drive relatively high expression levels in yeast, e.g.pPGK1, pTDH3, pADH1, pENO2. In another embodiment gRNA expression may bedriven by a pol II promoter known to drive mid-range expression levelsin yeast, e.g. pTEF1, pTEF2, pYEF3, pRPL3, pRPL15A. In yet anotherembodiment, gRNA expression may be drive by a pol II promoter known todrive relatively low expression levels in yeast, e.g. pRPL4, pSSB1,pSSA1, pPDA1, pCYC1. In other embodiments, the pol II promoter drivinggRNA expression may be selected from one of many constitutive promotersdriving expression in mammalian cells, including but not limited to,pCMV, pEF1a, pSV40, pPGK1, p Ubc, human beta actin promoter, pCAG, amongothers.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryoticcells can be yeast, fungi, algae, plant, animal, or human cells.Eukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, mouse, rat,rabbit, dog, or non-human mammals including non-human primates. Thechoice of nucleic acid-guided nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12a (e.g., CpfI), MAD2, or MAD7. As withthe guide nucleic acid, the nuclease may be encoded by a DNA sequence ona vector (e.g., the engine vector) and may be under the control of aninducible promoter.

In addition to the components described above, an editing cassette orribozyme-containing editing cassette may comprise one or more primersites. The primer sites can be used to amplify the editing cassettes orribozyme-containing editing cassettes and to assemble multiplexedediting cassettes or ribozyme-containing editing cassettes by usingoligonucleotide primers and bridging oligos; for example, if the primersites flank one or more of the other components of the editing cassette.

An editing cassette or ribozyme-containing editing cassette also maycomprise a barcode. A barcode is a unique DNA sequence that correspondsto the donor DNA sequence such that the barcode can identify the editmade to the corresponding target sequence. The barcode typicallycomprises four or more nucleotides. In some embodiments, the editingcassettes or ribozyme-containing editing cassettes comprise a collectionof donor nucleic acids representing, e.g., gene-wide or genome-widelibraries of donor nucleic acids. The library of editing cassettes orribozyme-containing editing cassettes is assembled into multiplexediting cassettes of at least two gRNA/donor DNA pairs and then clonedinto vector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, a vector encoding components of thenucleic acid-guided nuclease system further encodes a nucleicacid-guided nuclease comprising one or more nuclear localizationsequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs. In some embodiments, the engineered nucleasecomprises NLSs at or near the amino-terminus, NLSs at or near thecarboxy-terminus, or a combination.

The editing cassettes, ribozyme-containing editing cassettes, and/orvector backbones comprise control sequences operably linked to thecomponent sequences to be transcribed. As stated above, the pol IIpromoters driving transcription of one or more components of the nucleicacid-guided nuclease editing system (e.g., transcription of one or bothof the nuclease and gRNA) may be inducible. A number of gene regulationcontrol systems have been developed for the controlled expression ofgenes in plant, microbe, and animal cells, including mammalian cells,for example the pL promoter (induced by heat inactivation of the CI857repressor), the pBAD promoter (induced by the addition of arabinose tothe cell growth medium), and the rhamnose inducible promoter (induced bythe addition of rhamnose to the cell growth medium). Yeast induciblepromoters may be responsive to nutrient source, small molecule, hormoneresponse elements, nutrient depletion, or synthetic compounds. Yeastinducible promoter systems may include the PHO5 promoter, inducible bydepletion of inorganic phosphate (U.S. Pat. No. 4,880,734); the MET3promoter, suppressed by depletion of methionine (Mao, et al., CurrentMicrobiology, 45:37-40 (2002)); the CUP1 promoter, inducible by copper(U.S. Pat. No. 4,940,661); the GAL1 promoter, inducible by galactose andsuppressed by glucose (U.S. Pat. No. 5,139,936); or the GEV and ZEVsystems, engineered promoters responsive to estradiol induction (U.S.Pat. No. 9,212,359), among others.

FIG. 1A shows a simplified flow chart for exemplary method 100 forenriching for edited cells. Looking at FIG. 1A, method 100 begins bydesigning and synthesizing editing cassettes or ribozyme-containingediting cassettes 102. As described above, each editing cassette orribozyme-containing editing cassette comprises a gRNA sequence to betranscribed and a donor DNA sequence (e.g., homology arm sequence) to betranscribed comprising a desired target genome edits as well as a PAM orspacer mutation. In or ribozyme-containing editing cassettes, theediting cassette is linked to one or more sequences coding for aself-cleaving ribozyme. Once the editing cassettes orribozyme-containing editing cassettes have been synthesized, theindividual editing cassettes are amplified 104. The editing cassettes orribozyme-containing editing cassettes and linear vector backbones arethen used to transform cells 106 thereby creating a library oftransformed cells. The vector backbones typically comprise the codingsequence for a nuclease, as seen in FIG. 1F. Alternatively, the cellsmay already be expressing the nuclease (e.g., the cells may have alreadybeen transformed with a vector comprising the coding sequence for thenuclease or the coding sequence for the nuclease may be stablyintegrated into the cellular genome) such that only the vector backbonedoes not comprise a coding sequence for a nuclease.

A variety of delivery systems may be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell 108. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of particular interest is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat. No. 10,329,559,issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18 Jun. 2019; U.S.Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No. 10,465,185,issued 5 Nov. 2019; U.S. Pat. No. 10,519,437, issued 31 Dec. 2019; andU.S. Ser. No. 16/666,964, filed 29 Oct. 2019, and Ser. No. 16/680,643,filed 12 Nov. 2019 all of which are herein incorporated by reference intheir entirety. If the screening/selection module is one module in anautomated multi-module cell editing system, the cells are likelytransformed in an automated cell transformation module.

Once transformed 106, the cells can then be subjected to selection usingselection medium 108. Selectable markers and selection medium areemployed to select for cells that have received the vector backbone.Commonly used selectable markers include drug selectable markers such asampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin,tetracycline, gentamicin, bleomycin, streptomycin, puromycin,hygromycin, blasticidin, and G418.

Once cells that have been properly transformed are selected 108, thenext step in method 100 is to provide conditions for nucleic acid-guidednuclease editing 110. “Providing conditions” includes incubation of thecells in appropriate medium and may also include providing conditions toinduce transcription of an inducible promoter (e.g., adding antibiotics,increasing temperature) for transcription of one or both of thegRNA/donor DNA pair and nuclease. Once editing is complete, the cellsare allowed to recover and may be subjected to another round of editing112 or, alternatively, the cells may be used in research 114.

In certain embodiments of the present methods and compositions,expression of the editing cassette is driven by a pol II promoter and aself-cleaving ribozyme sequence is placed 3′ of the editing cassette.FIG. 1B depicts an exemplary editing cassette in the context of a pol IIpromoter and self-cleaving ribozyme. The pol II promoter (here, a pADH1promoter) is 5′ of the transcription start site and drives expression ofthe editing cassette. After the transcription start site, there is anediting cassette comprising a gRNA sequence (47 bp gRNA) that encodesboth the guide RNA that is complementary to the nuclease target sequenceand the gRNA scaffold sequence that complexes with the nuclease. Next,3′ of the gRNA sequence in the editing cassette is the donor DNAsequence (124 bp Donor DNA) that encodes the template for homologousrecombination including the desired edit and one or more PAM sitealterations. Although the gRNA sequence is shown 5′ to the donor DNA inthis editing cassette, the gRNA sequence may be 3′ to the donor DNAsequence as both the editing cassettes and or ribozyme-containingediting cassettes are agnostic regarding the order of the gRNA and donorDNA coding sequences. Following the donor DNA sequence is an HDVribozyme sequence that encodes the HDV-like self-cleaving ribozyme whichmediates cleavage of the poly(A) tail that is the result of pol IItranscription. Finally, a pol II terminator (Pol II Terminator)terminates pol II transcription of the editing cassette. Though notshown in FIG. 1B, a second self-cleaving ribozyme sequence may beincluded in this editing construct 3′ to the transcription start siteand 5′ to the coding sequence for the gRNA in the editing cassette.

FIG. 1C is depicts a dual or ribozyme-containing editing cassettearchitecture for achieving simultaneous combinatorial (e.g., multiplex)editing. The compositions and methods described herein for single editsusing a single or ribozyme-containing editing cassette architectureapply to multiple orribozyme-containing editing cassette architecture.For example—as with single-edit architectures-using a pol II promoter totranscribe multiple editing cassettes avoids potential terminationmotifs in the donor DNAs that would create an issue for pol IIItranscription systems. Additionally, the present pol II system enablesthe use of many synthetic pol II promoters that otherwise would not beavailable for gRNA expression. In addition, pol II promoters are lesssensitive to sequence changes than pol III promoters and enable use of arange of small molecule inducible promoters including the GAL1, GEV andZEV systems. As with the single edit ribozyme-containing editingcassette architecture, a dual or multiple ribozyme-containing editingcassette architecture employs a 3′ self-cleaving ribozyme-such as an HDVribozyme or hammerhead ribozyme—to cleave off the post-transcriptionallyadded polyA tail which is a nuclear export signal, thus increasing thelikelihood that the editing cassette transcript remains in the nucleus.However, in the exemplary embodiment shown in FIG. 1C, the dual ormultiple-cassette architecture also employs a 5′ self-cleaving ribozyme,in this case a hammerhead ribozyme, to remove the post-transcriptionallyadded 5′ cap which also increases the likelihood that the editingcassette is retained in the nucleus.

The dual editing cassette architecture shown in FIG. 1C comprises twogRNA/donor DNA pairs, a pol II promoter and two self-cleaving ribozymes.The pol II promoter (here, the constitutive pTEF1 promoter) is 5′ of thetranscription start site and drives transcription of the first andsecond self-cleaving peptides, first and second editing cassettes and alinker or spacer sequence. After the transcription start site, there isa coding sequence for a self-cleaving ribozyme, in this case ahammerhead ribozyme. The purpose of this self-cleaving ribozyme at the5′ end of the dual ribozyme-containing editing cassette—as statedabove—is to remove the post-transcriptionally added 5′ cap to the dualcassette transcript which promotes retaining the editing cassettes inthe nucleus. 3′ to the first self-cleaving ribozyme is a gRNA sequencethat encodes both the guide RNA that is complementary to the nucleasetarget sequence and the scaffold sequence which complexes with thenuclease. Following the first gRNA sequence in the orribozyme-containing editing cassette there is a first donor DNA sequence(here, coding for a, e.g., CAN1 or ADE2 knockout) that 1) encodes thetemplate for homologous recombination with the target sequence therebydirecting rational, precise edits to the target sequence, and 2)provides one or more edits to the target sequence that removes, mutates,or otherwise renders inactive a PAM or spacer region in the target.

The next element in the dual ribozyme-containing editing cassettearchitecture is an optional linker or spacer comprising a sequence thatpromotes cleavage between the two editing cassettes. The linker orspacer element may comprise 1) a tRNA sequence, which assists in theprocessing of the separate editing cassettes by exploiting theendogenous RNA processing sequences present in tRNAs; 2) an additionalself-cleaving ribozyme (such as a self-cleaving ribozyme in thehepatitis delta virus (HDV)-like ribozyme family, a self-cleavingribozyme in the glucosamine-6-phosphate synthase ribozyme family, aself-cleaving ribozyme in the hammerhead ribozyme family, aself-cleaving ribozyme in the hairpin ribozyme family, a self-cleavingribozyme in the Neurospora Varkud satellite ribozyme family, aself-cleaving ribozyme in the twister ribozyme family, a self-cleavingribozyme in the twister sister ribozyme family, a self-cleaving ribozymein the hatchet ribozyme family, or a self-cleaving ribozyme in thepistol ribozyme family); or 3) an exogenous cleavage factor recognitionsequence (such as Cys4).

Following the linker or spacer (e.g., 3′ of the linker) is the secondgRNA/donor DNA pair. The second gRNA/donor DNA pair comprises a secondgRNA sequence encoding the both the guide RNA that is complementary tothe nuclease target sequence and the scaffold sequence which complexeswith the nuclease; and a second donor DNA sequence (here, also a CAN1 orADE2 knockout) that encodes the template for homologous recombinationwith the target sequence comprising both the desired edit and one ormore PAM site alterations. 3′ to the second gRNA/donor DNA pair is asecond self-cleaving ribozyme sequence, here encoding the HDVself-cleaving ribozyme, which mediates cleavage of the poly(A) tail thatis the result of pol II transcription. Finally, 3′ to the HDV ribozymesequence is a pol II terminator which functions to terminate pol IItranscription of the dual editing cassette. Although in this FIG. 1C thegRNA is shown 5′ to the donor DNA in the gRNA/donor DNA pairs, in eitheror both gRNA/donor DNA pairs the gRNA may be 3′ of the donor DNA.Further, FIG. 1C shows two editing cassettes in the construct; however,there may be a third, fourth, fifth or sixth gRNA/donor DNA pair in theconstruct, wherein each of the a third, fourth, fifth or sixthgRNA/donor DNA pairs are separated by one another by a linker or spacersequence.

In addition to increased transcription and nuclear localization ofgRNAs, the present disclosure is drawn to increasing the efficiency ofnucleic acid-guided nuclease editing in yeast via multi-vectortransformations (MVTs). As described in detail above, in nucleicacid-guided nuclease genome editing, precise edits are created viahomology-directed repair of nuclease-mediated double strand breaks orsingle-strand nicks with a gRNA/donor DNA pair located on a plasmid. Thenumber of edits per cell is limited by the fact that, typically, onlyone plasmid containing one editing cassette is able to confer a singleedit in the genome of each cell transformed by an editing pool. Thepresent disclosure demonstrates that, via the gap repair assemblyprocess and adjusting the ratio of editing cassettes to vectorbackbones, individual yeast cells may be transformed by multiple vectorbackbones at once, thereby allowing the cell to be edited simultaneouslyby multiple editing cassettes contained within the vector backbones.This process is referred to herein as multi-vector transformation (MVT).

Multi-vector transformation occurs as a result of the gap repair plasmidcloning process, whereby a pool of linearized vector backbones—whereeach vector backbone comprises an antibiotic resistance gene or aportion of an antibiotic resistance gene as described infra—isco-transformed into yeast cells with a pool of editing cassettes thatcontain 1) a gRNA sequence, 2) a DNA donor sequence (preferablycomprising in addition to a sequence for a desired edit, a PAMmutation), and 3) sequences homologous to the linearized plasmidbackbone. Through the yeast cell's native homologous recombinationmachinery, the linearized plasmid backbone and editing cassette arejoined together into an editing plasmid, and the editing plasmid can beselected for via the antibiotic resistance gene.

MVT is a variation on the gap repair assembly process where multipleediting cassettes (or multiple ribozyme-containing editing cassettes)combine with linearized backbones (e.g., linearized backbones of thesame type or linearized backbones that comprise different antibioticresistance genes) to create more than one unique editing plasmid insideof the single yeast cell. The multiple unique editing plasmids aremaintained inside the single cell simultaneously due to the presence ofthe 2μ viral origin located on the editing plasmid, where the 2μ viralorigin of replication is the multi-copy origin, typically maintaining acopy number of roughly 50 plasmids—in this case editing plasmids—in anygiven cell.

Described herein are several ways in which multi-vector transformationediting can be optimized. First, by increasing the molar ratio of theediting cassette pool or ribozyme-containing editing cassette pool tothe linearized vector backbone, the number of different editing vectorsthat are assembled via gap repair and maintained in each cell isincreased. This embodiment is shown in FIG. 1D.

Second, the rate of MVT is increased when multiple linearized plasmidbackbones with different selection genes (e.g., antibiotic resistancegenes) are included in the transformation with the pool of editingcassettes or ribozyme-containing editing cassettes, followed byselection for yeast cells resistant to all antibiotic markers includedon the linearized plasmid backbones. This alternative embodiment isshown in FIG. 1E. In this embodiment, only cells that have beentransformed with linearized plasmid backbones with each of theselectable markers survive the selection thereby forcing multi-vectortransformation. It has been determined that the probability of a uniqueediting cassette or ribozyme-containing editing cassette becomingincorporated via gap repair into a plasmid backbone is a function of thesize of the editing cassette pool or ribozyme-containing editingcassette pool and the distribution of editing cassettes orribozyme-containing editing cassettes within the pool. The more editingcassettes or ribozyme-containing editing cassettes in the library andthe more uniform the pool of editing cassettes or ribozyme-containingediting cassettes, the higher the probability unique cassettes will beincorporated into the linearized plasmid backbones in each cell allowingfor multiple edits per cell.

Third, FIG. 1F(ii) shows an alternative or expansion of the method shownin FIG. 1E. In FIG. 1F(i), the method shown in FIG. 1E is shown inabbreviated form, where a library of editing cassettes are inserted intotwo different vector backbones comprising different antibioticresistance markers; however, the process of inserting the library ofediting cassette into the two different vector backbones is random. InFIG. 1F(ii), the different vector backbones not only comprise differentantibiotic resistance markers but also comprise different homologies forinserting the editing cassettes. That is, two different libraries ofediting cassettes are used, one library of editing cassettes withregions of homology to be inserted into vector backbone A and onelibrary of editing cassettes with regions of homology to be insertedinto vector backbone B. As described in more detail infra, such a schemecan prevent “collisions” between edits.

Finally in yet another alternative to the method shown in FIG. 1E,instead of using two different antibiotic resistance genes a singleantibiotic resistance gene is used, where one vector backbone codes fora first portion of the antibiotic resistance gene and a second vectorbackbone codes for a second portion of the antibiotic resistance gene;that is, a split antibiotic resistance gene is used. In such a system,the gene encoding an antibiotic resistance protein is split into two ormore segments and fused to inteins that can be rejoined by proteintrans-splicing. Each portion is carried on a vector backbone carrying anediting cassette. Delivery of the vector backbones to a population ofcells results in cells that comprise a partial set of portions of theantibiotic resistance gene or a full set of portions of the antibioticresistance genes. Only cells with a complete set of portions produce afully-reconstituted antibiotic resistance gene via protein splicing andthus only these cells can resist selection. This process is described inmore detail infra in relation to FIG. 1G.

Advantages of MVT editing include increasing the number of editspossible in each individual cell by placing selective pressure on eachcell to maintain more than one editing plasmid. Further, MVT editing maybe practiced with dual, triple or more editing cassettes orribozyme-containing editing cassettes; that is, use of editing cassettesor ribozyme-containing editing cassettes that comprise two or moregRNA/donor DNA pairs to further increase the number of edits per cellper round of editing.

FIG. 1D a simplified graphic of a MVT system for editing yeast genomes.In FIG. 1D, a pool (e.g., library) of editing cassettes is combined witha linear vector backbone comprising 1) a coding sequence for a nuclease,2) an antibiotic resistance gene, and 3) a 2μ origin of replication andthe editing cassettes and linear vector backbone are transformed intoyeast cells. Gap repair in the yeast cells inserts the editing cassettesinto the linear vector backbone via homologous recombination betweenhomologous sequences on the linear vector backbone and editing cassettethus creating an editing vector. Following transformation, cells thathave been properly transformed are selected by antibiotic selection,resulting in a library of cells comprising assembled editing vectors.Again, the probability of a unique editing cassette orribozyme-containing editing cassette becoming incorporated via gaprepair into a plasmid backbone is a function of the size of the editingcassette pool or ribozyme-containing editing cassette pool and thedistribution of editing cassettes or ribozyme-containing editingcassettes within the pool. The more editing cassettes orribozyme-containing editing cassettes in the library and the moreuniform the pool of editing cassettes or ribozyme-containing editingcassettes, the higher the probability unique cassettes will beincorporated into the linearized plasmid backbones in each cell allowingfor multiple edits per cell. Thus, the present embodiment uses asingle-type vector backbone but adjusting the size and uniformity of theediting cassette pool or ribozyme-containing editing cassette poolallows for multiple editing cassettes to be incorporated into vectorbackbones in each cell.

FIG. 1E is a simplified graphic of an alternative system for editingyeast genomes. In FIG. 1E, a pool (e.g., library) of editing cassettesor ribozyme-containing editing cassettes is combined with linear vectorbackbones comprising 1) a coding sequence for a nuclease, 2) anantibiotic resistance gene, and 3) a 2μ origin of replication. Theediting cassettes or ribozyme-containing editing cassettes and linearvector backbone are then transformed into yeast cells. The differencebetween the processes in FIG. 1D and FIG. 1E is that in FIG. 1E, atleast two different linear vector backbones are used, where thedifference between the two vector backbones is that one vector backbonecomprises a first antibiotic resistance gene (e.g., selection marker)and the other vector backbone comprises a second antibiotic resistancegene, and the first and second antibiotic resistance genes aredifferent. In FIG. 1E, gap repair in the yeast cells inserts the editingcassettes or ribozyme-containing editing cassettes (e.g., library ofediting cassettes or ribozyme-containing editing cassettes) into thelinear vector backbones via homologous recombination between homologoussequences on the linear vector backbone and editing cassette orribozyme-containing editing cassette to form editing vectors.

Following transformation, cells that have been properly transformed areselected by resistance to both the first and second antibiotics,resulting in a library of cells comprising assembled editing vectors.Because selective pressure on the cells requires the cells to take upand maintain editing vectors with both the first and second antibiotics,the cells are very likely to be transformed with two or more differentediting vectors, and thus two or more gRNA/donor DNA pairs (e.g.,editing sequences). Further, the 2μ origin of replication maintains eachvector or plasmid in the yeast cell at approximately 50 copies. Notethat here two different linear vector backbones with two differentantibiotic markers are used; however, three, four, or five differentlinear vector backbones with three, four or five different antibioticmarkers may be employed in the methods described herein. Thus, thisalternative method, in addition to adjusting the size and uniformity ofthe editing cassette pool or ribozyme-containing editing cassette poolallowing for multiple editing cassettes to be incorporated into vectorbackbones in each cell, adds the concept of selective pressure to assuremultiple vectors are maintained within each cell.

In some embodiments, the backbone to insert molar ratio is roughly threecassettes for each backbone, which in practice amounts to 500 ng ofbackbone and 50 ng of editing cassettes. In the present methods, thecassette concentration is increased up to 8×, as shown in FIG. 15 , andcan be as high as 50×, or as high as 40×, or as high as 30×, or as highas 20×. The library size may range from as few as two different editingcassettes or ribozyme-containing editing cassettes up to 100,000 editingcassettes or ribozyme-containing editing cassettes or more.

FIG. 1F(i) is a simplified graphic showing the method of FIG. 1E inshorthand form. Note that there is a single library of editing cassettesand two different vector backbones where the vector backbones comprisedifferent antibiotic resistance genes (Abx1 and Abx2). In this scheme,the editing cassettes from the library randomly are inserted into eithervector backbone Abx1 or vector backbone Abx2. FIG. 1F(ii) is a “twist”on the method shown in FIG. 1E and FIG. 1F(i). Here, instead of onelibrary of editing cassettes there are two libraries of editingcassettes where each library of editing cassettes where one library ofediting cassettes has regions of homology to be inserted into vectorbackbone Abx1 and one library of editing cassettes has regions ofhomology to be inserted into vector backbone Abx2 by gap repair. In thisscheme, editing cassettes are not randomly inserted into a single vectorbackbone, but may be selectively inserted into one of two, one of three,one of four, or more vector backbones where each different vectorbackbone comprises a different antibiotic resistance gene (or portionthereof as described infra). One advantage of this “twist” is that onemay avoid “collision control” and experience less “edit incompatibility”between editing cassettes. For example, when performing saturationmutagenesis of a region in a genome, if a single vector backbone is usedone editing cassette may cause genomic changes that inactivate thetarget regions for other edits to be made. However, if editing cassetteswith predicted incompatibility are in the same library and thus areinserted into the same vector backbone (e.g., vector backbone Abx1), itis unlikely incompatible editing cassettes will be transformed andmaintained in the same cell. That is, because of the selective pressureof two antibiotics in the edited cells, it is less likely that a cellwill be transformed with and maintain two editing vectors with the sameantibiotic resistance gene; instead, the cell will likely maintain twoediting vectors with different antibiotic resistance genes, hence twoediting cassettes that are compatible. Splitting the pool of editingcassettes in an intelligent manner between multiple vector backbonesincreases the likelihood that only “compatible” editing cassettes willbe present in a single cell.

FIG. 1G is a simplified graphic of an alternative system for editingyeast genomes related closely to the method described FIG. 1F(ii). InFIG. 1G, two pools (e.g., libraries) of editing cassettes orribozyme-containing editing cassettes—where each library of editingcassettes comprises a different homology region for being inserted intoa vector backbone—is combined with two different linear vectorbackbones. The first linear vector backbone comprises 1) a codingsequence for a nuclease, 2) a first portion of an antibiotic resistancegene fused to an N-terminal intein, 3) a homology insert regioncompatible with editing library 1, and 4) a 2μ origin of replication;and the second linear vector backbone comprises 1) a coding sequence fora nuclease, 2) a second portion of an antibiotic resistance gene fusedto a C-terminal intein, 3) a homology insert region compatible withediting library 2, and 4) a 2μ origin of replication. In thisembodiment, the coding region for a single antibiotic resistance gene isthus separated into portions, which is advantageous in that there is alimited number of antibiotics and antibiotic resistance genes known.Using inteins, the coding sequence for an antibiotic resistance gene canbe broken up into two, three or more parts instead of using two, threeor more different antibiotic resistance genes. Using a combination ofinteins and multiple antibiotic resistance genes many unique editingvectors can be used to confer multiple unique edits.

The editing cassettes or ribozyme-containing editing cassettes andlinear vector backbones are then transformed into yeast cells. Whereasthe method depicted in FIG. 1E uses at least two different linear vectorbackbones where each comprises a different antibiotic resistance gene,here the different vector backbones comprise portions of a singleantibiotic resistance gene fused to inteins. Inteins are self-splicingpolypeptides with an ability to excise themselves from flanking proteinregions with remarkable precision. (See, e.g., Pavankumar,Microorganisms, 6, 19 (2018) and Jillette, et al., NatureCommunications, (2019) 10:4968, both of which are incorporated byreference for all purposes.) Two well-known and well-characterizedinteins are those derived from Nostoc punctiforme PCC73102 split alphasubunit of the DNA polymerase III intein (NpuDnaE) and fromSynechocystis sp. PCC6803 DnaB helicase SspDnaB. In the process ofexcision, the inteins ligate the flanked host protein fragments. As inthe previously methods (e.g., depicted in FIGS. 1D, 1E, and 1F(ii)), gaprepair in the yeast cells inserts the editing cassettes orribozyme-containing editing cassettes (e.g., libraries of editingcassettes or ribozyme-containing editing cassettes) into the linearvector backbones via homologous recombination between homologoussequences on the linear vector backbone and editing cassette orribozyme-containing editing cassette to form editing vectors.

Following transformation, cells that have been properly transformed withall portions of the antibiotic resistance gene (here, in this FIG. 1Gthere are two) are selected by resistance to the single antibiotic. Theportions of the antibiotic resistance gene are transcribed andtranslated and the fused N-terminal and C-terminal inteins have theability to splice themselves out from the flanking protein fragments(exteins) post-transcriptionally, acting much like introns. Becauseselective pressure on the cells requires the cells to take up andmaintain editing vectors with both the first and second portions of thesingle antibiotic resistance gene, the cells are very likely to betransformed with both editing vectors, and thus two or more gRNA/donorDNA pairs (e.g., editing sequences). Again, the 2μ origin of replicationmaintains each vector or plasmid in the yeast cell maintains the vectorsat approximately 50 copies.

Note that in this example, two different linear vector backbones withtwo portions of a single antibiotic resistance gene are used; however,the antibiotic resistance gene may be split into three, four, or fiveportions fused to different N-terminal and C-terminal inteins;alternatively, e.g., four different linear vector backbones may be usedwith the first vector backbone comprising, e.g., a first portion of anAbx1 resistant gene fused to the N-terminus of intein1 (and a firsthomology for editing cassette inserts); the second vector backbonecomprising a second portion of an Abx1 resistant gene fused to theC-terminus of intein1 (and a second homology for editing cassetteinserts); the third vector backbone comprising a first portion of anAbx2 resistant gene fused to the N-terminus of intein2 (and a thirdhomology for editing cassette inserts); and the fourth vector backbonecomprising a second portion of an Abx2 resistant gene fused to theC-terminus of intein2 (and a fourth homology for editing cassetteinserts). Like the method depicted in FIG. 1E, this alternativemethod—in addition to adjusting the size and uniformity of the editingcassette pool or ribozyme-containing editing cassette pool allowing formultiple editing cassettes to be incorporated into vector backbones ineach cell—adds the concept of selective pressure to assure multiplevectors are maintained within each cell.

FIG. 1H is an exemplary editing vector map after gap repair, where theediting vector map comprises, inter alia, an editing cassette orribozyme-containing editing cassette (or compound editing cassette orribozyme-containing editing cassette), a selectable marker, and thecoding sequence for the nuclease MAD7. In the vector map in FIG. 1F, asingle editing cassette is shown. Beginning at 11:55 o'clock, there is apromoter driving transcription of the gRNA/donor DNA pair, and in someembodiments this promoter is a pol II promoter, followed by a terminatorsuch as a pol II or SUP4 terminator; a promoter driving transcription ofan antibiotic resistance gene 1 or 2 (chosen from at least two differentantibiotic resistance genes) or two different portions of a singleantibiotic resistance gene followed by a terminator; another promoterdriving transcription of an SV40 nuclear localization sequence and theMAD7 nuclease coding sequence followed by a terminator; a promoterdriving an ampicillin resistance gene (which is in a reverse orientationto the transcription of the other elements); a pUC origin of replicationfor propagation of the editing vector in bacteria; and a 2-μ origin ofreplication for propagation of multiple copies of each editing vector inyeast. Again, it should be apparent to one of ordinary skill in the artgiven the present disclosure that there may be more than one gRNA/donorDNA pair in the editing cassette or ribozyme-containing editingcassette; that is—for a two-edit editing cassette or ribozyme-containingediting cassette—there may be a promoter driving transcription of afirst gRNA, a first donor DNA sequence, followed by a second gRNA, and asecond donor DNA sequence followed by a terminator. In addition, thereoptionally may be penta-T or penta-T+G motifs between the gRNA/donor DNApairs; that is between the first gRNA/donor DNA sequence and the secondgRNA/donor DNA sequence.

In each of the different methods and compositions herein it should beapparent to one of ordinary skill in the art given the presentdisclosure that the methods and compositions provide for multiple editsper round per cell by using a compound editing cassette in a singlevector backbone or by using multiple vectors each with a differentediting cassette as well as performing multiple rounds of editing (e.g.,recursive editing) using these methods and compositions.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease Editing in Cells

Automated Cell Editing Instruments

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument to, e.g., perform one of the exemplary novel methods usingthe novel compositions described herein. The instrument, for example,may be and preferably is designed as a stand-alone desktop instrumentfor use within a laboratory environment. The instrument 200 (not labeledin this FIG. 2A, but see FIG. 2B) may incorporate a mixture of reusableand disposable components for performing the various integratedprocesses in conducting automated genome cleavage and/or editing incells without human intervention. Illustrated is a gantry 202, providingan automated mechanical motion system (actuator) (not shown) thatsupplies XYZ axis motion control to, e.g., an automated (i.e., robotic)liquid handling system 258 including, e.g., an air displacement pipettor232 which allows for cell processing among multiple modules withouthuman intervention. In some automated multi-module cell processinginstruments, the air displacement pipettor 232 is moved by gantry 202and the various modules and reagent cartridges remain stationary;however, in other embodiments, the liquid handling system 258 may staystationary while the various modules and reagent cartridges are moved.Also included in the automated multi-module cell processing instrument200 are reagent cartridges 210 comprising reservoirs 212 andtransformation module 230 (e.g., a flow-through electroporation deviceas described in detail in relation to FIGS. 5B-5F), as well as washreservoirs 206, cell input reservoir 251 and cell output reservoir 253.The wash reservoirs 206 may be configured to accommodate large tubes,for example, wash solutions, or solutions that are used often throughoutan iterative process. Although two of the reagent cartridges 210comprise a wash reservoir 206 in FIG. 2A, the wash reservoirs insteadcould be included in a wash cartridge where the reagent and washcartridges are separate cartridges. In such a case, the reagentcartridge 210 and a wash cartridge may be identical except for theconsumables (reagents or other components contained within the variousinserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. Also shownis pipette tip box 214 with pipette tips 215. In some examples, therobotic handling system 258 may include an automated liquid handlingsystem such as those manufactured by Tecan Group Ltd. of Mannedorf,Switzerland, Hamilton Company of Reno, Nev. (see, e.g., WO2018015544A1),or Beckman Coulter, Inc. of Fort Collins, Colo. (see, e.g.,US20160018427A1). Pipette tips may be provided in a pipette transfer tipsupply (not shown) for use with the air displacement pipettor 232.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises a cell growth vial 218 (described ingreater detail below in relation to FIGS. 3A-3D). Additionally seen isthe TFF module 222 (described above in detail in relation to FIGS.4A-4E). Also illustrated as part of the automated multi-module cellprocessing instrument 200 of FIG. 2A is a singulation module 240 (e.g.,a solid wall isolation, incubation and normalization device (SWIINdevice) is shown here) described herein in relation to FIGS. 6C-6F,served by, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Additionally seen is a selection module 220. Also note theplacement of three heatsinks 255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232. The deckof the multi-module cell processing instrument 200 may include aprotection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 200 are containedwithin a lip of the protection sink. Also seen are reagent cartridges210, which are shown disposed with thermal assemblies 211 which cancreate temperature zones appropriate for different regions, and pipettetip box 214. Note that one of the reagent cartridges also comprises aflow-through electroporation device 230 (FTEP), served by FTEP interface(e.g., manifold arm) and actuator 231. Also seen is TFF module 222 withadjacent thermal assembly 225, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 223. Thermal assemblies 225,235, and 245 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The rotating growth vial 218 iswithin a growth module 234, where the growth module is served by twothermal assemblies 235. Selection module is seen at 220. Also seen isthe SWIIN module 240, comprising a SWIIN cartridge 240, where the SWIINmodule also comprises a thermal assembly 245, cover 244, illumination243 (in this embodiment, backlighting), evaporation and condensationcontrol 249, and where the SWIIN module is served by SWIIN interface(e.g., manifold arm) and actuator 247. Also seen in this view is touchscreen display 201, display actuator 203, illumination 205 (one oneither side of multi-module cell processing instrument 200), and cameras239 (one illumination device on either side of multi-module cellprocessing instrument 200). Finally, element 237 comprises electronics,such as circuit control boards, high-voltage amplifiers, power supplies,and power entry; as well as pneumatics, such as pumps, valves andsensors.

FIG. 2C illustrates a front perspective view of multi-module cellprocessing instrument 200 for use in as a desktop version of theautomated multi-module cell editing instrument 200. For example, achassis 290 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 290 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 290 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 2C, chassis 290 includestouch screen display 201, cooling grate 264, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 200 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis290 is lifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270a-270 c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, forexample, allow for additional air flow beneath the chassis 290 (foot 270d is not shown).

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device, arotating growth vial 218 in a cell growth module 234, a tangential flowfiltration module 222, a SWIIN module 240 as well as interfaces andactuators for the various modules. In addition, chassis 290 housescontrol circuitry, liquid handling tubes, air pump controls, valves,sensors, thermal assemblies (e.g., heating and cooling units) and othercontrol mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat.No. 10,329,559, issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18Jun. 2019; U.S. Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No.10,465,185, issued 5 Nov. 2019; U.S. Pat. No. 10,519,437, issued 31 Dec.2019; and U.S. Ser. No. 16/680,643, filed 12 Nov. 2019; Ser. No.16/666,964, filed 29 Oct. 2019; Ser. No. 16/750,369, filed 23 Jan. 2020,all of which are herein incorporated by reference in their entirety.

The Rotating Cell Growth Module

FIG. 3A shows one embodiment of a rotating growth vial 300 for use withthe cell growth device and in the automated multi-module cell processinginstruments described herein. The rotating growth vial 300 is anoptically-transparent container having an open end 304 for receivingliquid media and cells, a central vial region 306 that defines theprimary container for growing cells, a tapered-to-constricted region 318defining at least one light path 310, a closed end 316, and a driveengagement mechanism 312. The rotating growth vial 300 has a centrallongitudinal axis 320 around which the vial rotates, and the light path310 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 310 is positioned in the lower constricted portion ofthe tapered-to-constricted region 318. Optionally, some embodiments ofthe rotating growth vial 300 have a second light path 308 in the taperedregion of the tapered-to-constricted region 318. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 310 is shorter than the second light path 308 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 308 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 312 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 312 such that the rotating growth vial 300 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 300 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 300 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 300 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 300 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 300 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 304with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 304 mayoptionally include an extended lip 302 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 300may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 300 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 300 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 300 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 400. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 300 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 3B is a perspective view of one embodiment of a cell growth device330. FIG. 3C depicts a cut-away view of the cell growth device 330 fromFIG. 3B. In both figures, the rotating growth vial 300 is seenpositioned inside a main housing 336 with the extended lip 302 of therotating growth vial 300 extending above the main housing 336.Additionally, end housings 352, a lower housing 332 and flanges 334 areindicated in both figures. Flanges 334 are used to attach the cellgrowth device 330 to heating/cooling means or other structure (notshown). FIG. 3C depicts additional detail. In FIG. 3C, upper bearing 342and lower bearing 340 are shown positioned within main housing 336.Upper bearing 342 and lower bearing 340 support the vertical load ofrotating growth vial 300. Lower housing 332 contains the drive motor338. The cell growth device 330 of FIG. 3C comprises two light paths: aprimary light path 344, and a secondary light path 350. Light path 344corresponds to light path 310 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 300, andlight path 350 corresponds to light path 308 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 316. Lightpaths 310 and 308 are not shown in FIG. 3C but may be seen in FIG. 3A.In addition to light paths 344 and 340, there is an emission board 348to illuminate the light path(s), and detector board 346 to detect thelight after the light travels through the cell culture liquid in therotating growth vial 300.

The motor 338 engages with drive mechanism 312 and is used to rotate therotating growth vial 300. In some embodiments, motor 338 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 338 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 336, end housings 352 and lower housing 332 of the cellgrowth device 330 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 300 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 330 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 330 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 330—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 330, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 3D illustrates a cell growth device 330 as part of an assembly 360comprising the cell growth device 330 of FIG. 3B coupled to light source390, detector 392, and thermal components 394. The rotating growth vial300 is inserted into the cell growth device. Components of the lightsource 390 and detector 392 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 332 that houses the motor that rotatesthe rotating growth vial 300 is illustrated, as is one of the flanges334 that secures the cell growth device 330 to the assembly. Also, thethermal components 394 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 330 to the thermal components 394 via the flange 334 on the baseof the lower housing 332. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 300 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 300 by piercing though the foil seal orfilm. The programmed software of the cell growth device 330 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 300. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 300 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 330 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 330 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 330 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional details regarding rotating growth vials and cell growthdevices see U.S. Pat. No. 10,435,662, issued 8 Oct. 2019; U.S. Pat. No.10,443,031, issued 15 Oct. 2019; and U.S. Ser. No. 16/552,981, filed 27Aug. 2019 and Ser. No. 16/780,640, filed 3 Feb. 2020.

The Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems to perform themethods described herein is a module or component that can grow, performbuffer exchange, and/or concentrate cells and render them competent sothat they may be transformed or transfected with the nucleic acidsneeded for engineering or editing the cell's genome.

FIG. 4A shows a retentate member 422 (top), permeate member 420 (middle)and a tangential flow assembly 410 (bottom) comprising the retentatemember 422, membrane 424 (not seen in FIG. 4A), and permeate member 420(also not seen). In FIG. 4A, retentate member 422 comprises a tangentialflow channel 402, which has a serpentine configuration that initiates atone lower corner of retentate member 422 specifically at retentate port428—traverses across and up then down and across retentate member 422,ending in the other lower corner of retentate member 422 at a secondretentate port 428. Also seen on retentate member 422 are energydirectors 491, which circumscribe the region where a membrane or filter(not seen in this FIG. 4A) is seated, as well as interdigitate betweenareas of channel 402. Energy directors 491 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 422 with permeate/filtrate member 420 via the energy directorcomponent 491 on permeate/filtrate member 420 (at right). Additionally,countersinks 423 can be seen, two on the bottom one at the top middle ofretentate member 422. Countersinks 423 are used to couple and tangentialflow assembly 410 to a reservoir assembly (not seen in this FIG. 4A butsee FIG. 4B).

Permeate/filtrate member 420 is seen in the middle of FIG. 4A andcomprises, in addition to energy director 491, through-holes forretentate ports 428 at each bottom corner (which mate with thethrough-holes for retentate ports 428 at the bottom corners of retentatemember 422), as well as a tangential flow channel 402 and twopermeate/filtrate ports 426 positioned at the top and center of permeatemember 420. The tangential flow channel 402 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 420 also comprisescountersinks 423, coincident with the countersinks 423 on retentatemember 420.

On the left of FIG. 4A is a tangential flow assembly 410 comprising theretentate member 422 and permeate member 420 seen in this FIG. 4A. Inthis view, retentate member 422 is “on top” of the view, a membrane (notseen in this view of the assembly) would be adjacent and under retentatemember 422 and permeate member 420 (also not seen in this view of theassembly) is adjacent to and beneath the membrane. Again countersinks423 are seen, where the countersinks in the retentate member 422 and thepermeate member 420 are coincident and configured to mate with threadsor mating elements for the countersinks disposed on a reservoir assembly(not seen in FIG. 4A but see FIG. 4B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 m, however for othercell types, the pore sizes can be as high as 20 μm. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 402 may vary depending on the volumeof the cell culture to be grown and the optical density of the cellculture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 m wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, orfrom 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400μm to 600 μm high. If the cross section of the flow channel 102 isgenerally round, oval or elliptical, the radius of the channel may befrom about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μmin hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, orfrom 300 m to 600 μm wide in hydraulic radius, or from about 200 to 500μm in hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 420 members may be different depending on thedepth of the channel in each member.

FIG. 4B shows front perspective (right) and rear perspective (left)views of a reservoir assembly 450 configured to be used with thetangential flow assembly 410 seen in FIG. 4A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 450that is coupled to the tangential flow assembly 410 seen in FIG. 4A) areretentate reservoirs 452 on either side of permeate reservoir 454. Alsoseen are permeate ports 426, retentate ports 428, and three threads ormating elements 425 for countersinks 423 (countersinks 423 not seen inthis FIG. 4B). Threads or mating elements 425 for countersinks 423 areconfigured to mate or couple the tangential flow assembly 410 (seen inFIG. 4A) to reservoir assembly 450. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 410 to reservoir assembly 450. In additionis seen gasket 445 covering the top of reservoir assembly 450. Gasket445 is described in detail in relation to FIG. 4E and one reservoir hasa pipette tip 405 inserted therein. At left in FIG. 4B is a rearperspective view of reservoir assembly 450, where “rear” is the side ofreservoir assembly 450 that is not coupled to the tangential flowassembly. Seen are retentate reservoirs 452, permeate reservoir 454,gasket 445, and one reservoir has a pipette tip 405 inserted therein.

The TFF device may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 4C depicts a top-down view of the reservoir assemblies 450 shown inFIG. 4B. FIG. 4D depicts a cover 444 for reservoir assembly 450 shown inFIGS. 4B and 4E depicts a gasket 445 that in operation is disposed oncover 444 of reservoir assemblies 450 shown in FIG. 4B. FIG. 4C is atop-down view of reservoir assembly 450, showing the tops of the tworetentate reservoirs 452, one on either side of permeate reservoir 454.Also seen are grooves 432 that will mate with a pneumatic port (notshown), and fluid channels 434 that reside at the bottom of retentatereservoirs 452, which fluidically couple the retentate reservoirs 452with the retentate ports 428 (not shown), via the through-holes for theretentate ports in permeate member 420 and membrane 424 (also notshown). FIG. 4D depicts a cover 444 that is configured to be disposedupon the top of reservoir assembly 450. Cover 444 has round cut-outs atthe top of retentate reservoirs 452 and permeate/filtrate reservoir 454.Again, at the bottom of retentate reservoirs 452 fluid channels 434 canbe seen, where fluid channels 434 fluidically couple retentatereservoirs 452 with the retentate ports 428 (not shown). Also shown arethree pneumatic ports 430 for each retentate reservoir 452 andpermeate/filtrate reservoir 454. FIG. 4E depicts a gasket 445 that isconfigures to be disposed upon the cover 444 of reservoir assembly 450.Seen are three fluid transfer ports 442 for each retentate reservoir 452and for permeate/filtrate reservoir 454. Again, three pneumatic ports430, for each retentate reservoir 452 and for permeate/filtratereservoir 454, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 426, collecting the cell culture through asecond retentate port 404 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 422)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 420) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 426.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall work flow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 420) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 404, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 426. All types of prokaryotic and eukaryoticcells-both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM,DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media maybe provided in a reagent cartridge as part of a kit. For culture ofadherent cells, cells may be disposed on beads, microcarriers, or othertype of scaffold suspended in medium. Most normal mammaliantissue-derived cells—except those derived from the hematopoieticsystem—are anchorage dependent and need a surface or cell culturesupport for normal proliferation. In the rotating growth vial describedherein, microcarrier technology is leveraged. Microcarriers ofparticular use typically have a diameter of 100-300 μm and have adensity slightly greater than that of the culture medium (thusfacilitating an easy separation of cells and medium for, e.g., mediumexchange) yet the density must also be sufficiently low to allowcomplete suspension of the carriers at a minimum stirring rate in orderto avoid hydrodynamic damage to the cells. Many different types ofmicrocarriers are available, and different microcarriers are optimizedfor different types of cells. There are positively charged carriers,such as Cytodex 1 (dextran-based, GE Healthcare), DE-52(cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based,Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- orECM-(extracellular matrix) coated carriers, such as Cytodex 3(dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4(polystyrene-based, Thermo Scientific); non-charged carriers, likeHyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based ongelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GEHealthcare).

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 404 while collecting the medium in one of thepermeate/filtrate ports 426 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeatee ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 404 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 426 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 404 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 404 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 404 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port426 on the opposite end of the device/module from the permeate port 426that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 62/728,365, filed 7 Sep. 2018; 62/857,599,filed 5 Jun. 2019; and 62/867,415, filed 27 Jun. 2019.

The Cell Transformation Module

FIG. 5A depicts an exemplary combination reagent cartridge andelectroporation device (“cartridge”) that may be used in an automatedmulti-module cell processing instrument along with the TFF module. Inaddition, in certain embodiments the material used to fabricate thecartridge is thermally-conductive, as in certain embodiments thecartridge contacts a thermal device (not shown), such as a Peltierdevice or thermoelectric cooler, that heats or cools reagents in thereagent reservoirs or reservoirs 510, 513, 521, 525. Reagent reservoirsor reservoirs 510, 513, 521, 525 may be reservoirs into which individualtubes 504 of reagents are inserted as shown in FIG. 5A, or the reagentreservoirs may hold the reagents without inserted tubes. Additionally,the reservoirs 510, 513, 521, 525 in a reagent cartridge may beconfigured for any combination of tubes, co-joined tubes, anddirect-fill of reagents.

In one embodiment, the reagent reservoirs or reservoirs 504 of reagentcartridge are configured to hold various size tubes, including, e.g.,250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorf ormicrocentrifuge tubes. In yet another embodiment, all reservoirs may beconfigured to hold the same size tube, e.g., 5 ml tubes, and reservoirinserts may be used to accommodate smaller tubes in the reagentreservoir. In yet another embodiment—particularly in an embodiment wherethe reagent cartridge is disposable the reagent reservoirs hold reagentswithout inserted tubes. In this disposable embodiment, the reagentcartridge may be part of a kit, where the reagent cartridge ispre-filled with reagents and the receptacles or reservoirs sealed with,e.g., foil, heat seal acrylic or the like and presented to a consumerwhere the reagent cartridge can then be used in an automatedmulti-module cell processing instrument. As one of ordinary skill in theart will appreciate given the present disclosure, the reagents containedin the reagent cartridge will vary depending on work flow; that is, thereagents will vary depending on the processes to which the cells aresubjected in the automated multi-module cell processing instrument,e.g., protein production, cell transformation and culture, cell editing,etc.

Reagents such as cell samples, enzymes, buffers, nucleic acid vectors,expression cassettes, proteins or peptides, reaction components (suchas, e.g., MgCl₂, dNTPs, nucleic acid assembly reagents, gap repairreagents, and the like), wash solutions, ethanol, and magnetic beads fornucleic acid purification and isolation, etc. may be positioned in thereagent cartridge at a known position. In some embodiments of thecartridge, the cartridge comprises a script (not shown) readable by aprocessor (not shown) for dispensing the reagents. Also, the cartridgeas one component in an automated multi-module cell processing instrumentmay comprise a script specifying two, three, four, five, ten or moreprocesses to be performed by the automated multi-module cell processinginstrument. In certain embodiments, the reagent cartridge is disposableand is pre-packaged with reagents tailored to performing specific cellprocessing protocols, e.g., genome editing or protein production.Because the reagent cartridge contents vary while components/modules ofthe automated multi-module cell processing instrument or system may not,the script associated with a particular reagent cartridge matches thereagents used and cell processes performed. Thus, e.g., reagentcartridges may be pre-packaged with reagents for genome editing and ascript that specifies the process steps for performing genome editing inan automated multi-module cell processing instrument, or, e.g., reagentsfor protein expression and a script that specifies the process steps forperforming protein expression in an automated multi-module cellprocessing instrument.

For example, the reagent cartridge may comprise a script to pipettecompetent cells from a reservoir, transfer the cells to a transformationmodule, pipette a nucleic acid solution comprising a vector withexpression cassette from another reservoir in the reagent cartridge,transfer the nucleic acid solution to the transformation module,initiate the transformation process for a specified time, then move thetransformed cells to yet another reservoir in the reagent cassette or toanother module such as a cell growth module in the automatedmulti-module cell processing instrument. In another example, the reagentcartridge may comprise a script to transfer a nucleic acid solutioncomprising a vector from a reservoir in the reagent cassette, nucleicacid solution comprising editing oligonucleotide cassettes in areservoir in the reagent cassette, and a nucleic acid assembly mix fromanother reservoir to the nucleic acid assembly/desalting module, ifpresent. The script may also specify process steps performed by othermodules in the automated multi-module cell processing instrument. Forexample, the script may specify that the nucleic acid assembly/desaltingreservoir be heated to 50° C. for 30 min to generate an assembledproduct; and desalting and resuspension of the assembled product viamagnetic bead-based nucleic acid purification involving a series ofpipette transfers and mixing of magnetic beads, ethanol wash, andbuffer.

As described in relation to FIGS. 5B and 5C below, the exemplary reagentcartridges for use in the automated multi-module cell processinginstruments may include one or more electroporation devices, preferablyflow-through electroporation (FTEP) devices. In yet other embodiments,the reagent cartridge is separate from the transformation module.Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. Applications of electroporationinclude the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies,drugs or other substances to a variety of cells such as mammalian cells(including human cells), plant cells, archea, yeasts, other eukaryoticcells, bacteria, and other cell types. Electrical stimulation may alsobe used for cell fusion in the production of hybridomas or other fusedcells. During a typical electroporation procedure, cells are suspendedin a buffer or medium that is favorable for cell survival. For bacterialcell electroporation, low conductance mediums, such as water, glycerolsolutions and the like, are often used to reduce the heat production bytransient high current. In traditional electroporation devices, thecells and material to be electroporated into the cells (collectively“the cell sample”) are placed in a cuvette embedded with two flatelectrodes for electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength; however, the flow-through electroporation devices included inthe reagent cartridges achieve high efficiency cell electroporation withlow toxicity. The reagent cartridges of the disclosure allow forparticularly easy integration with robotic liquid handlinginstrumentation that is typically used in automated instruments andsystems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

FIGS. 5B and 5C are top perspective and bottom perspective views,respectively, of an exemplary FTEP device 550 that may be part of (e.g.,a component in) the reagent cartridge in FIG. 5A or may be a stand-alonemodule; that is, not a part of a reagent cartridge or other module. FIG.5B depicts an FTEP device 550. The FTEP device 550 has wells that definecell sample inlets 552 and cell sample outlets 554. FIG. 5C is a bottomperspective view of the FTEP device 550 of FIG. 5B. An inlet well 552and an outlet well 554 can be seen in this view. Also seen in FIG. 5Care the bottom of an inlet 562 corresponding to well 552, the bottom ofan outlet 564 corresponding to the outlet well 554, the bottom of adefined flow channel 566 and the bottom of two electrodes 568 on eitherside of flow channel 566. The FTEP devices may comprise push-pullpneumatic means to allow multi-pass electroporation procedures; that is,cells to electroporated may be “pulled” from the inlet toward the outletfor one pass of electroporation, then be “pushed” from the outlet end ofthe FTEP device toward the inlet end to pass between the electrodesagain for another pass of electroporation. Further, this process may berepeated one to many times. For additional information regarding FTEPdevices, see, e.g., U.S. Pat. No. 10,435,713, issued 8 Oct. 2019; U.S.Pat. No. 10,443,074, issued 15 Oct. 2019; U.S. Pat. No. 10,323,258,issued 18 Jun. 2019; U.S. Pat. No. 10,508,288, issued 17 Dec. 2019; U.S.Pat. No. 10,415,058, issued 17 Sep. 2019; and U.S. Ser. No. 16/550,790,filed 26 Aug. 2019; and Ser. No. 16/571,080, filed 14 Sep. 2019.Further, other embodiments of the reagent cartridge may provide oraccommodate electroporation devices that are not configured as FTEPdevices, such as those described in U.S. Ser. No. 16/109,156, filed 22Aug. 2018. For reagent cartridges useful in the present automatedmulti-module cell processing instruments, see, e.g., U.S. Pat. No.10,376,889, issued 13 Aug. 2019; U.S. Pat. No. 10,406,525, issued 10Sep. 2019; U.S. Pat. No. 10,478,822, issued 19 Nov. 2019; U.S. Pat. No.10,576,474, issued 3 Feb. 2020; and U.S. Ser. No. 16/749,757, filed 22Jan. 2020.

Additional details of the FTEP devices are illustrated in FIGS. 5D-5F.Note that in the FTEP devices in FIGS. 5D-5F the electrodes are placedsuch that a first electrode is placed between an inlet and a narrowedregion of the flow channel, and the second electrode is placed betweenthe narrowed region of the flow channel and an outlet. FIG. 5D shows atop planar view of an FTEP device 550 having an inlet 552 forintroducing a fluid containing cells and exogenous material into FTEPdevice 550 and an outlet 554 for removing the transformed cells from theFTEP following electroporation. The electrodes 568 are introducedthrough channels (not shown) in the device. FIG. 5E shows a cutaway viewfrom the top of the FTEP device 550, with the inlet 552, outlet 554, andelectrodes 568 positioned with respect to a flow channel 566. FIG. 5Fshows a side cutaway view of FTEP device 550 with the inlet 552 andinlet channel 572, and outlet 554 and outlet channel 574. The electrodes568 are positioned in electrode channels 576 so that they are in fluidcommunication with the flow channel 566, but not directly in the path ofthe cells traveling through the flow channel 566. Note that the firstelectrode is placed between the inlet and the narrowed region of theflow channel, and the second electrode is placed between the narrowedregion of the flow channel and the outlet. The electrodes 568 in thisaspect of the device are positioned in the electrode channels 576 whichare generally perpendicular to the flow channel 566 such that the fluidcontaining the cells and exogenous material flows from the inlet channel572 through the flow channel 566 to the outlet channel 574, and in theprocess fluid flows into the electrode channels 576 to be in contactwith the electrodes 568. In this aspect, the inlet channel, outletchannel and electrode channels all originate from the same planar sideof the device. In certain aspects, however, the electrodes may beintroduced from a different planar side of the FTEP device than theinlet and outlet channels.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit. Alternatively, the FTEP device may also be formed in two or moreparallel layers, e.g., a layer with the horizontal channel and filter, alayer with the vertical channels, and a layer with the inlet and outletports, which are manufactured and/or molded individually and assembledfollowing manufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 508 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

As mentioned, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,where the flow channel decreases in width, the flow channel may narrowto between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μmand 2 mm, or between 75 μm and 1 mm. The distance between the electrodesin the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall sizeof the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cmin length, or 4.5 cm to 10 cm in length. The overall width of the FTEPdevice may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cmto 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is wide enough so thatat least two cells can fit in the narrowed portion side-by-side. Forexample, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

Cell Singulation and Enrichment Device

FIG. 6A depicts a solid wall device 6050 and a workflow for singulatingcells in microwells in the solid wall device. At the top left of thefigure (i), there is depicted solid wall device 6050 with microwells6052. A section 6054 of substrate 6050 is shown at (ii), also depictingmicrowells 6052. At (iii), a side cross-section of solid wall device6050 is shown, and microwells 6052 have been loaded, where, in thisembodiment, Poisson or substantial Poisson loading has taken place; thatis, each microwell has one or no cells, and the likelihood that any onemicrowell has more than one cell is low. At (iv), workflow 6040 isillustrated where substrate 6050 having microwells 6052 shows microwells6056 with one cell per microwell, microwells 6057 with no cells in themicrowells, and one microwell 6060 with two cells in the microwell. Instep 6051, the cells in the microwells are allowed to doubleapproximately 2-150 times to form clonal colonies (v), then editing isallowed to occur 6053. Shown are microwells 6057 where no cells weredistributed in a well and wells 6056 where a single cell was distributedin the well.

After editing 6053, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 6058),where cells that do not undergo editing thrive (microwells 6059) (vi).All cells are allowed to continue grow 6055 to establish colonies andnormalize, where the colonies of edited cells in microwells 6058 catchup in size and/or cell number with the cells in microwells 6059 that donot undergo editing (vii). Once the cell colonies are normalized, eitherpooling 6060 of all cells in the microwells can take place, in whichcase the cells are enriched for edited cells by eliminating the biasfrom non-editing cells and fitness effects from editing; alternatively,colony growth in the microwells is monitored after editing, and slowgrowing colonies (e.g., the cells in microwells 6058) are identified andselected 6061 (e.g., “cherry picked”) resulting in even greaterenrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for yeast cell growth includes LB, SOC, TPD, YPG, YPAD,MEM and DMEM.

A module useful for performing the method depicted in FIG. 6A is a solidwall isolation, incubation, and normalization (SWIIN) module. FIG. 6Bdepicts an embodiment of a SWIIN module 650 from an exploded topperspective view. In SWIIN module 650 the retentate member is formed onthe bottom of a top of a SWIIN module component and the permeate memberis formed on the top of the bottom of a SWIIN module component.

The SWIIN module 650 in FIG. 6B comprises from the top down, a reservoirgasket or cover 658, a retentate member 604 (where a retentate flowchannel cannot be seen in this FIG. 6B), a perforated member 601 swagedwith a filter (filter not seen in FIG. 6B), a permeate member 608comprising integrated reservoirs (permeate reservoirs 652 and retentatereservoirs 654), and two reservoir seals 662, which seal the bottom ofpermeate reservoirs 652 and retentate reservoirs 654. A permeate channel660 a can be seen disposed on the top of permeate member 608, defined bya raised portion 676 of serpentine channel 660 a, and ultrasonic tabs664 (not shown in this FIG. 6B, but see FIG. 6C) can be seen disposed onthe top of permeate member 608 as well. The perforations that form thewells on perforated member 601 are not seen in this FIG. 6B; however,through-holes 666 to accommodate the ultrasonic tabs 664 are seen. Inaddition, supports 670 are disposed at either end of SWIIN module 650 tosupport SWIIN module 650 and to elevate permeate member 608 andretentate member 604 above reservoirs 652 and 654 to minimize bubbles orair entering the fluid path from the permeate reservoir to serpentinechannel 660 a or the fluid path from the retentate reservoir toserpentine channel 660 b (neither fluid path is seen in this FIG. 6B).

In this FIG. 6B, it can be seen that the serpentine channel 660 a thatis disposed on the top of permeate member 608 traverses permeate member608 for most of the length of permeate member 608 except for the portionof permeate member 608 that comprises permeate reservoirs 652 andretentate reservoirs 654 and for most of the width of permeate member608. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 660 a (seen) and the counterpart 660 b (not seen butwould be disposed on the bottom of retentate member 604) can haveapproximately the same volume or a different volume. For example, each“side” or portion 660 a, 660 b of the serpentine channel may have avolume of, e.g., 2 mL, or serpentine channel 660 a of permeate member608 may have a volume of 2 mL, and the serpentine channel 660 b ofretentate member 604 may have a volume of, e.g., 3 mL. The volume offluid in the serpentine channel may range from about 2 mL to about 80mL, or about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL(note these volumes apply to a SWIIN module comprising a, e.g., 50-500Kperforation member). The volume of the reservoirs may range from 5 mL to50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or from 10 mL to 20mL, and the volumes of all reservoirs may be the same or the volumes ofthe reservoirs may differ (e.g., the volume of the permeate reservoirsis greater than that of the retentate reservoirs).

The serpentine channel portions 660 a (seen) and the counterpart 660 b(not seen but would be disposed on the bottom of retentate member 604)of the permeate member 608 and retentate member 604, respectively, areapproximately 200 mm long, 130 mm wide, and 4 mm thick, though in otherembodiments, the retentate and permeate members can be from 75 mm to 400mm in length, or from 100 mm to 300 mm in length, or from 150 mm to 250mm in length; from 50 mm to 250 mm in width, or from 75 mm to 200 mm inwidth, or from 100 mm to 150 mm in width; and from 2 mm to 15 mm inthickness, or from 4 mm to 10 mm in thickness, or from 5 mm to 8 mm inthickness. Embodiments the retentate (and permeate) members may befabricated from PMMA (poly(methyl methacrylate) or other materials maybe used, including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 6E and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400 ™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 650 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 650, or by applying atransparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g., FIG. 6E and the descriptionthereof infra.

In SWIIN module 650 cells and medium at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel660 b from ports in retentate member 604, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 660 a in permeate member 608. The cells are retained in themicrowells of perforated member 601 as the cells cannot travel throughthe filter. Appropriate medium may be introduced into permeate member608 through permeate ports (not seen). The medium flows upward throughfilter (not seen) to nourish the cells in the microwells (perforations)of perforated member 601. Additionally, buffer exchange can be effectedby cycling medium through the retentate and permeate members. Inoperation, the cells are deposited into the microwells, are grown for aninitial, e.g., 2-100 doublings, editing is induced by, e.g., raising thetemperature of the SWIIN to 42° C. to induce a temperature induciblepromoter or by removing growth medium from the permeate member andreplacing the growth medium with a medium comprising a chemicalcomponent that induces an inducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module650 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 660 a and thus to the filter (not seen) and pooled.Alternatively, if cherry picking is desired, the growth of the cellcolonies in the microwells is monitored, and slow-growing colonies aredirectly selected; or, fast-growing colonies are eliminated.

FIG. 6C is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 6C, it canbe seen that serpentine channel 660 a is disposed on the top of permeatemember 608 is defined by raised portions 676 and traverses permeatemember 608 for most of the length and width of permeate member 608except for the portion of permeate member 608 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 652can be seen). Moving from left to right, reservoir gasket 658 isdisposed upon the integrated reservoir cover 678 (cover not seen in thisFIG. 6C) of retentate member 604. Gasket 658 comprises reservoir accessapertures 632 a, 632 b, 632 c, and 632 d, as well as pneumatic ports 633a, 633 b, 633 c and 633 d. Also at the far left end is support 670.Disposed under permeate reservoir 652 can be seen one of two reservoirseals 662. In addition to the retentate member being in cross section,the perforated member 601 and filter (the filter is not seen in thisFIG. 6C) are in cross section. Note that there are a number ofultrasonic tabs 664 disposed at the right end of SWIIN module 650 and onraised portion 676 which defines the channel turns of serpentine channel660 a, including ultrasonic tabs 664 extending through through-holes 666of perforated member 601 (through-holes 666 are not seen in FIG. 6C butsee FIG. 6B). There is also a support 670 at the end distal reservoirs652, 654 of permeate member 608.

FIG. 6D is a side perspective view of an assembled SWIIIN module 650,including, from right to left, reservoir gasket 658 disposed uponintegrated reservoir cover 678 (not seen) of retentate member 604.Gasket 658 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 658 comprises reservoir access apertures 632 a, 632 b, 632 c, and632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d. Also atthe far-left end is support 670 of permeate member 608. In addition,permeate reservoir 652 can be seen, as well as one reservoir seal 662.At the far-right end is a second support 670.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 660.

FIG. 6E depicts the embodiment of the SWIIN module in FIGS. 6B-6Dfurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 698 comprises a SWIIN module 650seen lengthwise in cross section, where one permeate reservoir 652 isseen. Disposed immediately upon SWIIN module 650 is cover 694 anddisposed immediately below SWIIN module 650 is backlight 680, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 682, which is disposed over a heatsink 684. In thisFIG. 6E, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 686 and heat sink 688, as well as twothermoelectric coolers 692, and a controller 690 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability. For more detailsregarding solid wall isolation incubation and normalization devices seeU.S. Ser. No. 16/399,988, filed 30 Apr. 2019; Ser. No. 16/454,865, filed26 Jun. 2019; and Ser. No. 16/540,606, filed 14 Aug. 2019. Foralternative isolation, incubation and normalization modules, see U.S.Ser. No. 16/536,049, filed 8 Aug. 2019.

Use of the Automated Multi-Module Cell Processing Instrument

FIG. 7 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a bulkliquid growth module for induced editing and enrichment for editedcells. The cell processing instrument 700 may include a housing 744, areservoir of cells to be transformed or transfected 702, and a growthmodule (a cell growth device) 704. The cells to be transformed aretransferred from a reservoir to the growth module to be cultured untilthe cells hit a target OD. Once the cells hit the target OD, the growthmodule may cool or freeze the cells for later processing, or the cellsmay be transferred to a filtration/concentration module 730 where thecells are rendered electrocompetent and concentrated to a volume optimalfor cell transformation. Once concentrated, the cells are thentransferred to an electroporation device 708 (e.g.,transformation/transfection module).

In addition to the reservoir for storing the cells, the system 700 mayinclude a reservoir for storing editing cassettes or ribozyme-containingediting cassettes 716 and a vector backbone 718. Both the editingcassettes or ribozyme-containing editing cassettes and the vectorbackbone are transferred from the reagent cartridge to, e.g., anelectroporation device 708, which already contains the cell culturegrown to a target OD and rendered electrocompetent via filtration module730. In electroporation device 708, the assembled nucleic acids areintroduced into the cells. Following electroporation, the cells aretransferred into a combined recovery/selection module 710. For examplesof multi-module cell editing instruments, see U.S. Pat. No. 10,253,316,issued 9 Apr. 2019; U.S. Pat. No. 10,329,559, issued 25 Jun. 2019; U.S.Pat. No. 10,323,242, issued 18 Jun. 2019; U.S. Pat. No. 10,421,959,issued 24 Sep. 2019; U.S. Pat. No. 10,465,185, issued 5 Nov. 2019; U.S.Pat. No. 10,519,437, issued 31 Dec. 2019; and U.S. Ser. No. 16/680,643,filed 12 Nov. 2019; Ser. No. 16/666,964, filed 29 Oct. 2019; Ser. No.16/750,369, filed 23 Jan. 2020, all of which are herein incorporated byreference in their entirety.

Following recovery, and, optionally, selection 710, the cells aretransferred to a growth, and editing module 740. The cells are allowedto grow and editing take place. In some embodiments, editing is inducedby transcription of one or both of the nuclease and the gRNA being underthe control of an inducible promoter. In some embodiments, the induciblepromoter is a pL promoter where the promoter is activated by a rise intemperature and “deactivated” by lowering the temperature.

The recovery, selection, growth, editing and storage modules may all beseparate, may be arranged and combined as shown in FIG. 2A, or may bearranged or combined in other configurations. In certain embodiments,recovery and selection are performed in one module, and growth andediting are performed in a separate module. Alternatively, recovery,selection, growth, editing, and re-growth are performed in a singlemodule.

Once the cells are edited and re-grown (e.g., recovered from editing),the cells may be stored, e.g., in a storage module 712, where the cellscan be kept at, e.g., 4° C. until the cells are retrieved 714 forfurther study. Alternatively, the cells may be used in another round ofediting. The multi-module cell processing instrument is controlled by aprocessor 742 configured to operate the instrument based on user input,as directed by one or more scripts, or as a combination of user input ora script. The processor 742 may control the timing, duration,temperature, and operations of the various modules of the system 700 andthe dispensing of reagents. For example, the processor 742 may cool thecells post-transformation until editing is desired, upon which time thetemperature may be raised to a temperature conducive of genome editingand cell growth. The processor may be programmed with standard protocolparameters from which a user may select, a user may specify one or moreparameters manually or one or more scripts associated with the reagentcartridge may specify one or more operations and/or reaction parameters.In addition, the processor may notify the user (e.g., via an applicationto a smart phone or other device) that the cells have reached the targetOD as well as update the user as to the progress of the cells in thevarious modules in the multi-module system.

The automated multi-module cell processing instrument 700 is anuclease-directed genome editing system and can be used in singleediting systems where, e.g., two or more edits to a cellular genome areintroduced using a single editing process via multiplex editingcassettes. The system may be configured to perform sequential editing,e.g., using different nuclease-directed systems sequentially to providetwo or more genome edits in a cell in each of two or more rounds ofediting; and/or recursive editing, e.g. utilizing a singlenuclease-directed system to introduce sequentially two or more genomeedits in a cell in each of two or more round of editing.

FIG. 8 illustrates another embodiment of a multi-module cell processinginstrument. This embodiment depicts an exemplary system that performsrecursive gene editing on a yeast cell population. The cell processinginstrument 800 may include a housing 826, a reservoir for storing cellsto be transformed or transfected 802, and a cell growth module(comprising, e.g., a rotating growth vial) 804. The cells to betransformed are transferred from a reservoir to the cell growth moduleto be cultured until the cells hit a target OD. Once the cells hit thetarget OD, the growth module may cool or freeze the cells for laterprocessing or transfer the cells to a cell concentration module 806where the cells are subjected to buffer exchange and renderedelectrocompetent, and the volume of the cells may be reducedsubstantially. Once the cells have been concentrated to an appropriatevolume, the cells are transferred to electroporation device 808. Inaddition to the reservoir for storing cells 812, the multi-module cellprocessing instrument includes a reservoir for storing the vectorbackbone and editing cassettes or ribozyme-containing editing cassettes822. The vector backbones and editing cassettes or ribozyme-containingediting cassettes are transferred to the electroporation device 808,which already contains the cell culture grown to a target OD. In theelectroporation device 808, the nucleic acids are electroporated intothe cells. Following electroporation, the cells are transferred into anoptional recovery and dilution module 810, where the cells recoverbriefly post-transformation.

After recovery, the cells may be transferred to a storage module 812,where the cells can be stored at, e.g., 4° C. for later processing orretrieved 814, or the cells may be diluted and transferred to aselection/singulation/growth/induction/editing/normalization (SWIIN)module 820. In the SWIIN 820, the cells are arrayed such that there isan average of one cell per microwell. The arrayed cells may be inselection medium to select for cells that have been transformed ortransfected with the editing vector(s). Once singulated, the cells growthrough 2-50 doublings and establish colonies. Once colonies areestablished, editing is induced by providing conditions (e.g.,temperature, addition of an inducing or repressing chemical) to induceediting. Editing is then initiated and allowed to proceed, the cells areallowed to grow to terminal size (e.g., normalization of the colonies)in the microwells and then are treated to conditions that cure theediting vector from this round. Once cured, the cells can be flushed outof the microwells and pooled, then transferred to the storage (orrecovery) unit 812 or can be transferred back to the growth module 804for another round of editing. In between pooling and transfer to agrowth module, there typically is one or more additional steps, such ascell recovery, medium exchange (rendering the cells electrocompetent),cell concentration (typically concurrently with medium exchange by,e.g., filtration. Note that theselection/singulation/growth/induction/editing/normalization and curingmodules may be the same module, where all processes are performed in,e.g., a solid wall device, or selection and/or dilution may take placein a separate vessel before the cells are transferred to the solid wallsingulation/growth/induction/editing/normalization/editing module(SWIIN). Similarly, the cells may be pooled after normalization,transferred to a separate vessel, and cured in the separate vessel. Asan alternative to singulation in, e.g., a solid wall device, thetransformed cells may be grown in—and editing can be induced in-bulkliquid. Once the putatively-edited cells are pooled, they may besubjected to another round of editing, beginning with growth, cellconcentration and treatment to render electrocompetent, andtransformation by yet another donor nucleic acid in another editingcassette via the electroporation module 808.

In electroporation device 808, the yeast cells selected from the firstround of editing are transformed by a second set of editing cassettes orribozyme-containing editing cassettes and vector backbones and the cycleis repeated until the cells have been transformed and edited by adesired number of, e.g., editing cassettes. The multi-module cellprocessing instrument exemplified in FIG. 8 is controlled by a processor824 configured to operate the instrument based on user input or iscontrolled by one or more scripts including at least one scriptassociated with the reagent cartridge. The processor 824 may control thetiming, duration, and temperature of various processes, the dispensingof reagents, and other operations of the various modules of theinstrument 800. For example, a script or the processor may control thedispensing of cells, reagents, vectors, and editing oligonucleotides;which editing oligonucleotides are used for cell editing and in whatorder; the time, temperature and other conditions used in the recoveryand expression module, the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor may be programmed to notify a user (e.g., via anapplication) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 7 or 8 , then the resulting edited culture may gothrough another (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing cassettes (orribozyme-containing editing cassettes). For example, the cells fromround 1 of editing may be diluted and an aliquot of the edited cellsedited by editing cassette A may be combined with editing cassette B, analiquot of the edited cells edited by editing cassette A may be combinedwith editing cassette C, an aliquot of the edited cells edited byediting cassette A may be combined with editing cassette D, and so onfor a second round of editing. After round two, an aliquot of each ofthe double-edited cells may be subjected to a third round of editing,where, e.g., aliquots of each of the AB-, AC-, AD-edited cells arecombined with additional editing cassettes, such as editing cassettes X,Y, and Z. That is that double-edited cells AB may be combined with andedited by editing cassettes X, Y, and Z to produce triple-edited editedcells ABX, ABY, and ABZ; double-edited cells AC may be combined with andedited by editing cassettes X, Y, and Z to produce triple-edited cellsACX, ACY, and ACZ; and double-edited cells AD may be combined with andedited by editing cassettes X, Y, and Z to produce triple-edited cellsADX, ADY, and ADZ, and so on. In this process, many permutations andcombinations of edits can be executed, leading to very diverse cellpopulations and cell libraries. That is, the methods and compositionsdisclosed herein allow for multi-vector transformation with 1) a libraryof backbones having the same selective marker or a library of backboneshaving more than one selective marker; and 2) a library of editingcassettes or ribozyme-containing editing cassette having a singlegRNA/donor DNA pair or a library of editing cassettes orribozyme-containing editing cassettes having two or more gRNA/donor DNApairs. These multi-vector transformations can then be performedsequentially any number of times to introduce many edits into each cell.

In any recursive process, it is advantageous to “cure” the editingvectors (e.g., the gap-repaired vector backbone+the editing cassette orribozyme-containing editing cassette). “Curing” is a process in whichone or more editing vectors used in the prior round of editing iseliminated from the transformed cells. Curing can be accomplished by,e.g., cleaving the editing vector(s) using a curing plasmid therebyrendering the editing vectors nonfunctional; diluting the editingvector(s) in the cell population via cell growth (that is, the moregrowth cycles the cells go through, the fewer daughter cells will retainthe editing vector(s)), or by, e.g., utilizing a heat-sensitive originof replication on the editing vector. The conditions for curing willdepend on the mechanism used for curing; that is, in this example, howthe curing plasmid cleaves the editing vector.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation: 5 nM of oligonucleotides synthesized on achip were amplified using Q5 polymerase in 50 μL volumes. The PCRconditions were 95° C. for 1 minute; 8 rounds of 95° C. for 30seconds/60° C. for 30 seconds/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. Following amplification, the PCR products weresubjected to SPRI cleanup, where 30 L SPRI mix was added to the 50 μLPCR reactions and incubated for 2 minutes. The tubes were subjected to amagnetic field for 2 minutes, the liquid was removed, and the beads werewashed 2× with 80% ethanol, allowing 1 minute between washes. After thefinal wash, the beads were allowed to dry for 2 minutes, 50 μL 0.5×TE pH8.0 was added to the tubes, and the beads were vortexed to mix. Theslurry was incubated at room temperature for 2 minutes, then subjectedto the magnetic field for 2 minutes. The eluate was removed and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation: A 10-fold serial dilution series of purifiedbackbone was performed, and each of the diluted backbone series wasamplified under the following conditions: 95° C. for 1 minute; then 30rounds of 95° C. for 30 seconds/60° C. for 1.5 minutes/72° C. for 2.5minutes; with a final hold at 72° C. for 5 minutes. After amplification,the amplified backbone was subjected to SPRI cleanup as described abovein relation to the cassettes. The backbone was eluted into 100 μL ddH₂Oand quantified before isothermal assembly.

Gibson Assembly: 150 ng backbone DNA was combined with 100 ng cassetteDNA. An equal volume of 2× Gibson Master Mix was added, and the reactionwas incubated for 45 minutes at 50° C. After assembly, the assembledbackbone and cassettes were subjected to SPRI cleanup, as describedabove.

Example 2: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was used togrow a yeast cell culture which was monitored in real time using anembodiment of the cell growth device described herein. The rotatinggrowth vial/cell growth device was used to measure OD₆₀₀ in real time ofyeast s288c cells in YPAD medium. The cells were grown at 30° C. usingoscillating rotation and employing a 2-paddle rotating growth vial. FIG.9 is a graph showing the results. Note that OD₆₀₀ 6.0 was reached in 14hours.

Example 3: Cell Concentration

The TFF module as described above in relation to FIGS. 4A-4E has beenused successfully to process and perform buffer exchange on yeastcultures. A yeast culture was initially concentrated to approximately 5ml using two passes through the TFF device in opposite directions. Thecells were washed with 50 ml of 1M sorbitol three times, with threepasses through the TFF device after each wash. After the third pass ofthe cells following the last wash with 1M sorbitol, the cells werepassed through the TFF device two times, wherein the yeast cell culturewas concentrated to approximately 525 μl. FIG. 10 presents the filterbuffer exchange performance for yeast cells determined by measuringfiltrate conductivity and filter processing time. Target conductivity(˜10 S/cm) was achieved in approximately 23 minutes utilizing three 50ml 1M sorbitol washes and three passes through the TFF device for eachwash. The volume of the cells was reduced from 20 ml to 525 μl. Recoveryof approximately 90% of the cells has been achieved.

Example 4: Production and Transformation of Electrocompetent S.Cerevisiae

For testing transformation of the FTEP device in yeast, S. Cerevisiaecells were created using the methods as generally set forth inBergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013). Briefly,YPAD media was inoculated for overnight growth, with 3 ml inoculate toproduce 100 ml of cells. Every 100 ml of culture processed resulted inapproximately 1 ml of competent cells. Cells were incubated at 30° C. ina shaking incubator until they reached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 11 . The device showed better transformation and survivalof electrocompetent S. Cerevisiae at 2.5 kV voltages as compared to theNEPAGENE™ method. Input is total number of cells that were processed.

Examples Singulation of Yeast Colonies in a Solid Wall Device

Electrocompetent yeast cells were transformed with a cloned library, anisothermal assembled library, or a process control sgRNA plasmid(escapee surrogate). Electrocompetent Saccharomyces cerevisiae cellswere prepared as follows: The afternoon before transformation was tooccur, 10 mL of YPAD was inoculated with the selected Saccharomycescerevisiae strain. The culture was shaken at 250 RPM and 30° C.overnight. The next day, 100 mL of YPAD was added to a 250-mL baffledflask and inoculated with the overnight culture (around 2 mL ofovernight culture) until the OD600 reading reached 0.3+/−0.05. Theculture was placed in the 30° C. incubator shaking at 250 RPM andallowed to grow for 4-5 hours, with the OD checked every hour. When theculture reached an OD600 of approximately 1.5, 50 mL volumes were pouredinto two 50-mL conical vials, then centrifuged at 4300 RPM for 2 minutesat room temperature. The supernatant was removed from all 50 ml conicaltubes, while avoiding disturbing the cell pellet. 50 mL of a LithiumAcetate/Dithiothreitol solution was added to each conical tube and thepellet was gently resuspended. Both suspensions were transferred to a250 mL flask and placed in the shaker; then shaken at 30° C. and 200 RPMfor 30 minutes.

After incubation was complete, the suspension was transferred to two50-mL conical vials. The suspensions then were centrifuge at 4300 RPMfor 3 minutes, then the supernatant was discarded. Following the lithiumacetate/Dithiothreitol treatment step, cold liquids were used and thecells were kept on ice until electroporation. 50 mL of 1 M sorbitol wasadded and the pellet was resuspended, then centrifuged at 4300 RPM, 3minutes, 4° C., after which the supernatant was discarded. The 1Msorbitol wash was repeated twice for a total of three washes. 50 uL of 1M sorbitol was added to one pellet, cells were resuspended, thentransferred to the other tube to suspend the second pellet. The volumeof the cell suspension was measured and brought to 1 mL with cold 1 Msorbitol. At this point the cells were electrocompetent and could betransformed with a cloned library, an isothermal assembled library, orprocess control sgRNA plasmids.

In brief, a required number of 2-mm gap electroporation cuvettes wereprepared by labeling the cuvettes and then chilling on ice. Theappropriate plasmid—or DNA mixture—was added to each correspondingcuvette and placed back on ice. 100 uL of electrocompetent cells wastransferred to each labelled cuvette, and each sample was electroporatedusing appropriate electroporator conditions. 900 uL of room temperatureYPAD Sorbitol media was then added to each cuvette. The cell suspensionwas transferred to a 14 ml culture tube and then shaken at 30° C., 250RPM for 3 hours. After a 3 hr recovery, 9 ml of YPAD containing theappropriate antibiotic, e.g., geneticin or Hygromycin B, was added. Atthis point the transformed cells were processed in parallel in the solidwall device and the standard plating protocol, so as to compare“normalization” in the sold wall device with the standard benchtopprocess. Immediately before cells the cells were introduced to thepermeable-bottom solid wall device, the 0.45 M filter forming the bottomof the microwells was treated with a 0.1% TWEEN™ (polysorbate 20, orIUPAC polyoxyethylene (20) sorbitan monolaurate) solution to effectproper spreading/distribution of the cells into the microwells of thesolid wall device. The filters were placed into a Swinnex Filter Holder(47 mm, Millipore®, SX0004700) and 3 ml of a solution with 0.85% NaCland 0.1% TWEEN™ (polysorbate 20, or IUPAC polyoxyethylene (20) sorbitanmonolaurate) was pulled through the solid wall device and filter throughusing a vacuum. Different TWEEN™ (polysorbate 20, or IUPACpolyoxyethylene (20) sorbitan monolaurate) concentrations wereevaluated, and it was determined that for a 47 mm diameter solid walldevice with a 0.45 M filter forming the bottom of the microwells, apre-treatment of the solid wall device+filter with 0.1% TWEEN™(polysorbate 20, or IUPAC polyoxyethylene (20) sorbitan monolaurate) waspreferred (data not shown).

After the 3-hour recovery in YPAD the transformed cells were diluted anda 3 ml volume of the diluted cells was processed through the TWEEN™(polysorbate 20, or IUPAC polyoxyethylene (20) sorbitanmonolaurate)-treated solid wall device and filter, again using a vacuum.The number of successfully transformed cells was expected to beapproximately 1.0E+06 to 1.0E+08, with the goal of loading approximately10,000 transformed cells into the current 47 mm permeable-bottom solidwall device (having ˜30,000 wells). Serial dilutions of 10⁻¹, 10⁻², and10⁻³ were prepared, then 100 μL volumes of each of these dilutions werecombined with 3 ml 0.85% NaCl, and the samples were loaded onto solidwall devices. Each permeable-bottom solid wall device was then removedfrom the Swinnex filter holder and transferred to an LB agar platecontaining carbenicillin (100 μg/ml), chloramphenicol (25 μg/ml) andarabinose (1% final concentration). The solid wall devices were placedmetal side “up,” so that the permeable-bottom membrane was touching thesurface of the agar such that the nutrients from the plate could travelup through the filter “bottom” of the wells. The solid wall devices onthe YPD agar plates were incubated for 2-3 days at 30° C.

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 ml of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereallowed cells to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 ml conical centrifuge tube. The OD600 of thecell suspension was measured; at this point the cells can be processedin different ways depending on the purpose of the study. For example, ifan ADE2 stop codon mutagenesis library is used, successfully-editedcells should result in colonies with a red color phenotype when theresuspended cells are spread onto YPD agar plates and allowed to growfor 4-7 days. This phenotypic difference allows for a quantification ofpercentage of edited cells and the extent of normalization of edited andunedited cells.

Example 6: Multiplex Editing of Yeast

FIG. 12 shows the results of multiplex editing of yeast using an RNA polII gRNA expression system. A 500-member library of editing cassettes,designed to created silent swap mutations in various CDSs in the yeastgenome was transformed into yeast along with a cassette backbonecontaining RNA pol II gRNA expression machinery, MAD7 expressionmachinery and a selectable drug marker, KanMX. Transformed cells wereselected for via antibiotic containing agar. Individual colonies fromthe agar were picked into selective liquid media grown overnight. DNAfrom the overnight cultures was then extracted and shotgun sequenced ona NextSeq. Genomic DNA from the extractions was aligned against theintended edited sequence of the cassette contained within the individualcolonies. A star on the plate indicates that the correct intended editwas found in the individual colony. A circle on the plate indicates theintended edit was not found in the genomic DNA.

FIG. 13 is a graph showing the edit rates for dual cassettearchitectures tested in yeast where knockout edits are made to each ofthe CAN1 and ADE2 genes. Here, a phenotypic readout was used. ADE2knockouts result in a red phenotype for the cell colonies and CAN1knockouts allow for growth on canavanine agar medium. The first set ofthree bars shows a 95% edit rate for the ADE2 gene, an 85% edit rate forthe CAN1 gene, and an 85% edit rate for both the CAN1 and ADE2 genes.These results correspond to a dual cassette architecturepTEF1-HH-ADE2-P2-CAN1-HDV, where “pTEF1” denotes the Pol II promoter,“HH” denotes the hammerhead ribozyme, “ADE2” denotes the ADE2 editingcassette comprising both the gRNA and donor DNA, “P2” denotes a primersequence, “CAN1” denotes the CAN1 editing cassette comprising both thegRNA and donor DNA, and “HDV” denotes the HDV ribozyme. The second setof three bars shows a 95% edit rate for the ADE2 gene, a 50% edit ratefor the CAN1 gene, and a 50% edit rate for both the CAN1 and ADE2 genes.These results correspond to the dual cassette architecturepTEF1-HH-ADE2-tRNA(ala)-CAN1-HDV, where “tRNA(ala)” denotes the tRNA foralanine. The third set of three bars shows a 90% edit rate for the ADE2gene, a 55% edit rate for the CAN1 gene, and a 50% edit rate for boththe CAN1 and ADE2 genes. The third set of three bars corresponds to thedual cassette architecture pTEF1-HH-ADE2-tRNA(gly)-CAN1-HDV, where“tRNA(gly)” denotes the tRNA for glycine. The fourth set of three barsshows a 90% edit rate for the ADE2 gene, a 75% edit rate for the CAN1gene, and a 70% edit rate for both the CAN1 and ADE2 genes. The fourthset of three bars corresponds to the dual cassette architecturepTEF1-HH-ADE2-tRNA(thr)-CAN1-HDV, where “tRNA(thr)” denotes the tRNA forthreonine. The last set of three bars shows a 95% edit rate for the CAN1gene, a 50% edit rate for the ADE2 gene and a 50% edit rate for both theCAN1 and ADE2 genes. The last set of three bars corresponds to the dualcassette architecture pTEF1-HH-CAN1-tRNA(gly)-ADE2-HDV, where“tRNA(gly)” denotes the tRNA for glycine. Thus, the dual editingcassette architecture described herein results in at least 50% of editedcells comprising both the ADE2 and CAN1 edits.

Example 7: Multiplex Simultaneous Editing in Yeast

In the methods used herein, electrocompetent S. cerevisiae cells weretransformed with a linear vector backbone and a library of editingcassettes. Electrocompetent Saccharomyces cerevisiae cells were preparedas follows: The afternoon before transformation was to occur, 10 mL ofYPAD was inoculated with the selected Saccharomyces cerevisiae strain.The culture was shaken at 250 RPM and 30° C. overnight. The next day,100 mL of YPAD was added to a 250-mL baffled flask and inoculated withthe overnight culture (around 2 mL of overnight culture) until the OD600reading reached 0.3+/−0.05. The culture was placed in the 30° C.incubator shaking at 250 RPM and allowed to grow for 4-5 hours, with theOD checked every hour. When the culture reached an OD600 ofapproximately 1.5, 50 mL volumes were poured into two 50-mL conicalvials, then centrifuged at 4300 RPM for 2 minutes at room temperature.The supernatant was removed from all 50 ml conical tubes, while avoidingdisturbing the cell pellet. 50 mL of a Lithium Acetate/D dithiothreitolsolution was added to each conical tube and the pellet was gentlyresuspended. Both suspensions were transferred to a 250 mL flask andplaced in the shaker; then shaken at 30° C. and 200 RPM for 30 minutes.

After incubation was complete, the suspension was transferred to two50-mL conical vials. The suspensions then were centrifuge at 4300 RPMfor 3 minutes, then the supernatant was discarded. Following the lithiumacetate/Dithiothreitol treatment step, cold liquids were used and thecells were kept on ice until electroporation. 50 mL of 1 M sorbitol wasadded and the pellet was resuspended, then centrifuged at 4300 RPM, 3minutes, 4° C., after which the supernatant was discarded. The 1Msorbitol wash was repeated twice for a total of three washes. 50 uL of 1M sorbitol was added to one pellet, cells were resuspended, thentransferred to the other tube to suspend the second pellet. The volumeof the cell suspension was measured and brought to 1 mL with cold 1 Msorbitol.

500 ng linear vector backbone and 50 ng of the editing cassette librarywere added to an electroporation cuvette and placed on ice. 100 μL ofthe electrocompetent cells were added to the cuvette and electroporatedunder the following conditions: poring pulse: 1800 V, 5.0 msec pulselength, 50.0 msec pulse interval, 1 pulse; transfer pulse: 100 V, 50.0msec pulse length, 50.0 msec pulse interval, and 3 pulses. Followingelectroporation, the cells were transferred to a 15 mL tube and shakenat 30° C. and 250 rpm for 3 hours. 9 mL of YPAD and 10 μL G418 1000×stock was added to the tube. 10 μL of each transformation dilution wasspread on 2XYPD+Kan plates and incubated at 30° C. for 3 days.

FIG. 14 shows the results for using the methods disclosed above to editvarious loci in the genome of S. cerevisiae. In the data generated forFIG. 14 , a library of 500 different editing cassettes were combinedwith a single linear vector backbone, and 9 random colonies were pickedfor analysis. Eight samples comprised two different edits, and onesample comprised three different edits. Note that the fraction of cellscomprising more than one edit ranged from 7 to 80%.

FIG. 15 shows the unique plasmids observed via NextGen sequencing of a500-member editing library across different editing cassette library DNAconcentrations. Note that at 1× (e.g., 50 ng of editing cassettelibrary) there was a mean of 0.9375 unique plasmids identified per well,but at 50×, a mean of 1.6875 unique plasmids were identified per well.

FIG. 16 shows two bar graphs demonstrating that forced MVT and dualantibiotic selection approximately doubles the dual edit rate comparedto single antibiotic selection. In the two bar graphs on the left, inthe left-most bar graph, 6% dual edits were observed, 33.2% GFP knockout edits were observed, 39.1% mCherry knock out edits were observed,and 21.3% were unedited; in the right bar graph in this duo, 10.2% dualedits were observed, 37.2% GFP knock out edits were observed, 27.1%mCherry knock out edits were observed, and 25.6% were unedited. In thetwo bar graphs on the right, in the left bar graph, 10.7% dual editswere observed, 33.2% GFP knock out edits were observed, 16.7% mCherryknock out edits were observed, and 39.3% were unedited; and in theright-most bar graph 24.7% dual edits were observed, 41.0% GFP knock outedits were observed, 16.1% mCherry knock out edits were observed, and18.2% were unedited;

Example 8: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument. lacZF172 functionally knocks out the lacZ gene. “lacZ_F172*” indicates thatthe edit happens at the 172nd residue in the lacZ amino acid sequence.Following assembly, the product was de-salted in the isothermal nucleicacid assembly module using AMPure beads, washed with 80% ethanol, andeluted in buffer. The assembled editing vector and recombineering-ready,electrocompetent E. Coli cells were transferred into a transformationmodule for electroporation. The transformation module comprised anADP-EPC cuvette. See, e.g., U.S. Pat No. 62/551,069. The cells andnucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The paramters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) and allowed to recover inSOC medium containing chloramphenicol. Carbenicillin was added to themedium after 1 hour, and the cells were allowed to recover for another 2hours. After recovery, the cells were held at 4° C. until recovered bythe user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 9: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. Thetransformation module comprised an ADP-EPC cuvette. The cells andnucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are snot to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. A method for RNA-directed nuclease editing in yeast cellscomprising the steps of: designing and synthesizing a first linearvector backbone library comprising a coding sequence for a nuclease, acoding sequence for a first portion of a first antibiotic resistancegene fused to an N-terminus of a first intein, first homology regionsfor inserting a first library of editing cassettes, and a yeast originof replication; designing and synthesizing a second linear vectorbackbone library comprising a coding sequence for a nuclease, a codingsequence for a second portion of a first antibiotic resistance genefused to an C-terminus of the first intein, second homology regions forinserting a second library of editing cassettes, and a yeast origin ofreplication; designing and synthesizing the first library of editingcassettes, wherein each editing cassette comprises a gRNA and a donorDNA, wherein the gRNAs and donor DNAs of different cassettes targetdifferent target regions in a yeast genome, and wherein homology existsbetween the first library of editing cassettes and the first linearvector library; designing and synthesizing the second library of editingcassettes, wherein each editing cassette comprises a gRNA and a donorDNA, wherein the gRNAs and donor DNAs of different cassettes targetdifferent target regions in a yeast genome, and wherein homology existsbetween the second library of editing cassettes and the second linearvector library; transforming the yeast cells with the first and secondlinear vector backbone libraries and first and second libraries ofediting cassettes; selecting for transformed yeast cells by selectingfor antibiotic resistance to the first antibiotic; and providingconditions for RNA-directed nuclease editing in the yeast cells toproduce first edited yeast cells.
 2. The method of claim 1, furthercomprising the steps of: designing and synthesizing a third linearvector backbone library comprising a coding sequence for a nuclease,third homology regions for inserting a third library of editingcassettes, a coding sequence for a first portion of a second antibioticresistance gene fused to the N-terminus of a second intein, and a yeastorigin of replication; designing and synthesizing a fourth linear vectorbackbone library comprising a coding sequence for a nuclease, fourthhomology regions for inserting a fourth library of editing cassettes, acoding sequence for a second portion of a second antibiotic resistancegene fused to a C-terminus of the second intein, and a yeast origin ofreplication; designing and synthesizing the third library of editingcassettes, wherein each editing cassette comprises a gRNA and a donorDNA, wherein the gRNAs and donor DNAs of different cassettes targetdifferent target regions in a yeast genome, and wherein homology existsbetween the third library of editing cassettes and the third linearvector library; designing and synthesizing the fourth library of editingcassettes, wherein each editing cassette comprises a gRNA and a donorDNA, wherein the gRNAs and donor DNAs of different cassettes targetdifferent target regions in a yeast genome, and wherein homology existsbetween the fourth library of editing cassettes and the fourth linearvector library; transforming the first edited yeast cells with the firstand second linear vector backbone libraries and first and secondlibraries of editing cassettes; selecting for transformed first editedyeast cells by selecting for antibiotic resistance to the secondantibiotic; and providing conditions for RNA-directed nuclease editingin the yeast cells to produce second edited yeast cells.
 3. The methodof claim 2, wherein the second intein is derived from Nostoc punctiformePCC73102 split alpha subunit of the DNA polymerase III intein (NpuDnaE),Synechocystis sp. PCC6803 DnaB helicase SspDnaB, or CfaDnaE.
 4. Themethod of claim 3, wherein the second intein is derived from Nostocpunctiforme PCC73102 split alpha subunit of the DNA polymerase IIIintein (NpuDnaE).
 5. The method of claim 3, wherein the second intein isderived from Synechocystis sp. PCC6803 DnaB helicase SspDnaB.
 6. Themethod of claim 3, wherein the second intein is derived from CfaDnaE. 7.The method of claim 2, wherein the first and second antibioticresistance genes confer resistance to hygromycin, G418, puromycin,blasticidin or nourseothricin and the first and second antibioticresistance genes are different.
 8. The method of claim 2, wherein eachlinear vector backbone in each third and fourth linear backbone libraryfurther comprises an origin of replication functional in bacteria
 9. Themethod of claim 1, wherein the first intein is derived from Nostocpunctiforme PCC73102 split alpha subunit of the DNA polymerase IIIintein (NpuDnaE), Synechocystis sp. PCC6803 DnaB helicase SspDnaB, orCfaDnaE.
 10. The method of claim 9, wherein the first intein is derivedfrom Nostoc punctiforme PCC73102 split alpha subunit of the DNApolymerase III intein (NpuDnaE).
 11. The method of claim 9, wherein thefirst intein is derived from Synechocystis sp. PCC6803 DnaB helicaseSspDnaB.
 12. The method of claim 9, wherein the second first is derivedfrom CfaDnaE.
 13. The method of claim 1, wherein the first antibioticresistance gene confers resistance to hygromycin, G418, puromycin,blasticidin or nourseothricin.
 14. The method of claim 1, wherein someof the editing cassettes of the first and second libraries of editingcassettes comprise two gRNAs and two donor DNAs.
 15. The method of claim14, some of the editing cassettes of the first and second libraries ofediting cassettes comprise three gRNAs and three donor DNAs.
 16. Themethod claim 1, where each linear vector backbone in each linearbackbone library further comprises a promoter driving expression of theediting cassette.
 17. The method of claim 16, wherein the promoter is apol II promoter.
 18. The method of claim 17, wherein the pol II promoteris a pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2, pYEF3, pRPL3,pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, or pPDA1 promoter.
 19. The methodof claim 1, wherein each linear vector backbone in each first and secondlinear backbone library further comprises an origin of replicationfunctional in bacteria.
 20. The method of claim 1, wherein the yeastcells are Saccharomyces cerevisiae cells.